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.

Hemostasis
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

BV Injury Neural
Tissue Factor

Blood Vessel
Constriction

Platelet
Activation

Coagulation
Activation

Primary hemostatic plug Reduced Blood flow

Stable Hemostatic Plug
Plt-Fusion

Thrombin,

Fibrin

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

Review: Vascular System

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

Review: Vascular System

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

Vasoconstriction
Initial response to vessel injury.
Short-lived

More pronounced in vessels with medial smooth muscles Dependent on local contraction of smooth muscle. Subsequently linked to platelet plug formation.

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

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. Endothelin-1 = synthesized by injured endothelium Serotonin (5-hydroxytryptamine) = made by platelet aggregation and endothelium Bradykinin, Fibrinopeptides

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 400,000/ 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. If not consumed in a clotting reaction, platelets are normally removed by the spleen.

Platelet Plug Formation

Contents of Platelets
Actin, 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

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 Willebrands 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

Phospholipids

Platelet Plug Formation

Once the platelets normal environment is changed, they become activated or adhesive Three stages of plug formation
Platelet activation and adhesion Platelet aggregation Platelet secretion and release

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. Exposure to surfaces in the tissues causes them to bind to collagen with the presence of von Willebrand factor ( vWF) and Glycoprotein IbIX, 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

Platelet Secretion and Release

Requires ATP Platelets release contents of their granules, causing vasoconstriction Granules trigger a secondary aggregation which is irreversible Granules consist of
Alpha granules: Factor V, Factor VIII:vWF, Fibrinogen, 2-antiplasmin, platelet factor 4 Dense bodies: ATP, ADP, serotonin, Ca

Platelet Secretion and Release

Granules consist of
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

Platelet Plug Formation

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

 Activation of Protein Kinase C leads to:

Phosphorylation of pleckstrin
Change of platelet shape Release of contents of the storage granules Aggregation

Platelet Aggregation

Summary and Overview

Vascular Endothelial Injury
Platelet hemostatic function Vasoconstriction Coagulation activation via tissue factor-factor VIIa IXa, Xa complexes on activated platelets Thrombin

Subendothelial Collagen
Platelet adhesion secretion
Reversible ADP, serotonin, Ca2+, fibrinogen

Platelet aggregation secretion

Irreversible ADP, serotonin, Ca2+, fibrinogen

Platelet aggregation

Fibrinogen
PLATELET-FIBRIN THROMBUS

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

o o o

o o o o
o o o

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

o o

Physical Properties Groupings

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

Blood Coagulation
Mechanism Three essential steps
Rupture of the vessel or damage to the blood a complex cascade of blood coagulation factors. 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 cells, and plasma to form the clot.

Blood Coagulation
Two Pathways
Extrinsic
Initiation of the fibrin clot in response to tissue injury. Damage to tissue outside the vessel

Intrinsic
Trauma to the blood itself Damage to the blood vessel (collagen exposed)

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

Bradykinin

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

Extrinsic Pathway
Tissue Factor
From liver

Phospholipids from the membranes of the tissue Lipoprotein complex as proteolytic enzyme

Extrinsic Pathway
From liver

-carboxyglutamate (Gla)-containing zymogens (II, VII, IX, and X), the Gla residues in the amino terminal serve as highaffinity binding sites for Ca2+.

Extrinsic Pathway
From liver

Tissue factor acts as a cofactor for factor VIIa. Tissue factor + factor VIIa = TISSUE FACTOR COMPLEX
Also activate factor IX in the intrinsic pathway.

Extrinsic Pathway
From liver

Extrinsic Tenase Complex (activates factor Xa)

Ca2+ Tissue factor complex Factor X

Assembly occurs on the platelets membrane.

 Prothrombinase Complex
Ca2+ Factor Va Factor Xa Prothrombin

Takes place on the membrane of an activated platelet

Has exposed acidic phospholipid phosphatidylserine.

Prothrombin to Thrombin

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

Fibrinogen to Fibrin

Consists of three nonidentical pairs of polypeptide chains (A, B, )2 covalently linked by disulfide bonds.
B, contains Asp-linked complex oligosaccharides

Fibrinogen to Fibrin

A chain = Fibrinopeptide A (FPA); B chain = Fibrinopeptide B (FPB)

Bear excess negative charges at the amino terminal ends of the chains as a result of the presence of Asp, Glu residues, and tyrosine-O-sulfate in FPB. Contribute to solubility Prevent aggregation

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

Fibrinogen to Fibrin

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 -amino groups of lysine residues. More stable fibrin clot

Fibrinogen to Fibrin

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

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, FXI and FXIII)

FVa

Prothrombinase

Thrombi types
White thrombus
Platelets + Fibrin; poor in erythrocytes Forms at the site of an injury or abnormal vessel wall, particularly in areas with rapid blood flow.

Red Thrombus
Erythrocytes + fibrin Resembles clot in test tube Forms within vessels with retarded blood flow or stasis, with or without injury.

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

Fibrinolysis
Plasmin Responsible for fibrin and fibrinogen degradation Serine protease Circulates as plasminogen
Activated plasminogen are inactivated by 2antiplasmin Binds to fibrin and is incorporated in clots

Fibrinolysis
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

Fibrinolysis
Tissue plasminogen activator (t-PA)
From vascular endothelium under injury/ stress. Catalytically inactive unless bound to fibrin.

Urokinase
Precursor: PROUROKINASE Originally isolated in urine Synthesized by monocytes, macrophages, fibroblasts, epithelial cells, etc. Action: degradation of ECM

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

Fibrinolysis

Balance of Hemostasis

Virchows Triad

Regulation of Thrombin
To prevent further fibrin formation or platelet activation. 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

Coagulation Inhibition System

Provides balance and control of clotting mechanisms Natural inhibitors and anticoagulants circulate in the plasma to:
Prevent clotting when its not needed Limit or localize the clotting that is needed