Hemostasis and Thrombosis Notes

Hemostasis and Thrombosis

• Integrated sequence: Renal and Cardiovascular- PHAR 563
• Dr. Assem

Hemostasis and Thrombosis Learning Objectives

• Understand roles of platelets, endothelial cells, and coagulation factors in hemostasis and thrombosis.
• Review blood coagulation cascade.
• Name pharmacologic classes and specific drugs.
• Describe mechanism of action, pharmacokinetics differences, side effects, contraindications, and drug interactions for major drugs.

Hemostasis and Thrombosis: Definition and Goal

• Hemo: Blood; Stasis: Stable, balanced, steady, not changing
• Regulated dynamic process of maintaining fluidity of the blood.
• Process to stop bleeding after blood vessels injuries.
  • Vasoconstriction
  • Platelet plug
  • Blood clot
• Goal:
  • Repair vascular injury.
  • Limit blood loss.
  • Avoid vessel occlusion (Thrombosis) and inadequate perfusion of vital organs.
• Thrombosis refers to the pathological formation of blood clots due to inappropriate activation of hemostatic mechanisms.

Hemostasis and Thrombosis: Epidemiology

• Responsible for: ischemic stroke, heart disease, myocardial infarctions, pulmonary embolism, peripheral gangrene, and more.
• Impact in worldwide morbidity and mortality with over 10 million deaths a year.
• \[> 600,000\] deaths a year in the US.
• 1 Million hospitalization a year in the US.
• Treatment most effective within 6 hours of onset of symptoms.
• Differences between arterial and venous blood vessels:
  • Arterial thrombosis: Ischemic necrosis (reduction of supply)
  • Venous thrombosis: Edema, inflammation, embolism (reduction of blood return to the lungs)

Hemostasis and Thrombosis: Platelets-Endothelial Cells Interactions

• Blood cells:
  • Red blood cells (erythrocytes): Oxygen
  • White blood cells (leukocytes): Immune defense
  • Platelets: Hemostasis
• Physiopathology:
  • Platelets: Primary hemostasis
  • Never in contact with endothelial cells
  • Upon injury or lesion:
    • Adhere to exposed reactive endothelial matrix
  • Coagulation cascade: Secondary hemostasis
    • All coagulation factors have an inactive and active (a) form
    • Once activated, factor will activate the next clotting factor in the cascade
    • End result: Transformation of soluble fibrinogen to insoluble fibrine by thrombin
  • Regulation of coagulation
    • Multiple coagulation factors, calcium, and endothelial factors

Hemostasis and Thrombosis: Clot Formation

• (a): Local vasoconstriction, due to local spasm of the smooth muscle
• (b): Formation of platelet aggregate (plug), platelets are attracted by ADP release from the vessel
• Platelets release of 5HT, ADP, and TXA2 which accelerate vasoconstriction
• (c): Formation of blood clot: Thrombin converts soluble fibrinogen into insoluble fibrin (mesh)

Endothelium Roles

• Modulates many aspects of hemostasis
• Injured endothelium: Major initiator of coagulation
• Roles:
  • 1- Antithrombotic
    • Antiplatelet (barrier, PGI2, and nitric oxide)
    • Anticoagulant (ATIII, thrombomodulin, TF inhibitor)
    • Fibrinolytic (tPA)
  • 2- Prothrombotic
    • vWF (Von Willebrand Factor)
    • TF (tissue factor)
    • Plasminogen activator inhibitors
  • 3- Vascular repair
    • Growth factors to stimulate smooth muscle growth, fibroblasts, angiogenesis… PDGF, FGF, and TGF-β

Platelets

• Anucleate cells with multiple granules and multiple organelles, derived from megakaryocytes
• Circulate as inactive, non-binding concave discs
• On stimulation, undergo major shape change
• Develop receptors for clotting factors
• Develop ability to bind to each other and to sub-endothelium
• Platelet life-span: 8-12 days; eliminated by spleen

Platelets Granules

• Platelet granules are of two types: alpha granules and dense bodies.
• Alpha granules contain Platelet Derived Growth Factor (PDGF), platelet factor 4, Factors V & XIII…
• Dense bodies contain serotonin, nucleotides (ADP, ATP), and calcium.

Platelets Cell Membrane

• The cell membrane of platelets contains:
  • Receptors for collagen, vessel wall von Willebrand factor, and fibrinogen
  • Phospholipids, that play an important role in blood clotting
• Platelets GpIIb/IIIa
  • Exposed Collagen
  • Endothelium vWF
  • COLLAGEN
  • Fibrinogen ➔ Aggregation Adhesion
  • ADP
  • Adrenaline
  • THROMBIN

Platelets in Coagulation: 4 Stages

• 1- Adhesion
  • Interaction between tissues factors (vWF) and platelets GPIb receptor (1-3 seconds)
  • This binding induces activation of platelets
• 2- Activation
  • Platelets release ADP and Thromboxane (TxA2), these help to recruit other platelets into the plug and as secondary hemostasis gets started thrombin is generated, causing more platelet stimulation and conversion of fibrinogen to fibrin (3-7 minutes)
• 3- Secretion
  • Platelets secrete alpha and dense granules content
  • Form mesh
• 4- Clot formation

Platelets Regulation

• Platelet function is regulated by:
  • 1- Catecholamines, collagen, thrombin, and prostacyclin: Generated by cells surrounding platelets and act on platelet membrane receptors
  • 2- ADP and serotonin: Generated by platelets
  • 3- Prostaglandin endoperoxides and thromboxane A2, cAMP, cGMP, and calcium: Generated by platelets
• All these are targeted by several drugs
• Platelets activation mechanisms

Arachidonic Acid Pathway

• Arachidonic acid is released from the platelet cell membrane by phospholipase and rapidly oxidized by the enzymes cyclo-oxygenases to PGG2 & PGH2
• In the platelets: PGG2 is converted to TX A2 a highly potent platelet aggregator and constrictor of arterial muscle
• In the arterial endothelium: PGG2 & PGH2 are converted to prostacyclin (PGI2) a potent inhibitor of platelet aggregation and a vasodilator

P2Y (ADP) Receptors Pathway

• Response to its activation is the inhibition of the production of cAMP, dephosphorylation of the vasodilator-stimulated phosphoprotein (VASP), and activation of the GTPase Rap1B and phosphoinositide 3-kinase (PI3-K).
• These processes lead to platelets aggregation and thrombus

Coagulation Cascade General Features

• Zymogens converted to enzymes by limited proteolysis
• Complex formation requiring calcium, phospholipid surface, cofactors
• Thrombin converts fibrinogen to fibrin monomer
• Fibrin monomer crosslinked into fibrin
• Forms "glue" for platelet plug

General features of Zymogens Factors: II, VII, IX, X, XI, XII

• ~200 AA residues at C-term end are very homologous to trypsin and contain the protease active site
• 9-12 glutamate residues near the N-term end are converted to carboxyglutamate residues during biosynthesis in the liver
• These glutamate residues bind calcium and are necessary for the coagulation process

Coagulation Factors

• Table 17.3 Blood Clotting Factors (Procoagulants)
  • Refer to the provided table for a detailed list of factors, their names, nature, source, and pathways/functions.
  • Note: Synthesis of Factors II, VII, IX, and X requires vitamin K.

Hemostasis Intrinsic and Extrinsic Pathways

• Intrinsic pathway: Clot in the vessels
• Extrinsic pathway: Tissue injury
• Coagulation mechanism is composed of an extrinsic and an intrinsic pathways, which eventually merge into one
• The complex sequence of events that produce fibrin are divided into three stages

Coagulation Stage I: Formation of Prothrombin Activator (Xa)

• Extrinsic pathway (contact activation pathway)
  • Blood in contact with injured endothelium – tissue thromboplastin (III) interacts with proconvertin (VII), and Ca2+ leading to activation of Stuart factor (X).

Coagulation Stage I: Formation of Prothrombin Activator (Xa)

• Intrinsic pathway (Tissue factor pathway)
  • Exposed collagen activates Hageman factor (XII).
  • XIIa activates plasma thromboplastin antecedent (PTA; XI) which in interacting with Ca 2+ activates Christmas factor (IX).
  • IXa interacts with antihemophilic factor (VIII) and Ca 2+ to form a complex that activates Stuart factor (X).

Coagulation Stage I: Formation of Prothrombin Activator (Xa)

• Common pathway:
  • Xa in the presence of Ca2+ forms complexes with accelerin (V) to form prothrombin activator

Coagulation Stage II: Conversion of Prothrombin to Thrombin (IIa)

• Prothrombin – inactive precursor of thrombin
• In the presence of prothrombin activator and Ca2+ , prothrombin (II) is converted to thrombin (IIa)
• Thrombin increases its own rate of formation (positive feedback mechanism)

Thrombin

• Serine protease
• Cleaves fibrinogen to fibrin
• Activates V to Va, VIII to VIIIa, and XIII to XIIIa
• Activates platelets- cleaves thrombin receptor
• In presence of thrombomodulin activates Protein C

Coagulation Stage III: Conversion of Fibrinogen (I) to Fibrin

• Fibrinogen – plasma protein produced by the liver
• Thrombin converts fibrinogen (I) to fibrin
• Thrombin also activates fibrin-stabilizing factor (XIII), which in the presence of Ca2+, stabilizes the fibrin polymer through covalent bonding of fibrin monomers

GP = glycoprotein. GPIIb/IIIa receptor pathway

• Schematic overview of action of glycoprotein IIb/IIIa antagonists.

Coagulation (Non-Protein Factors)

• Calcium ions ($Ca^{2+}$)
  • Calcium required for promotion and acceleration of almost all blood clotting reactions
  • Except: activation of XII and XI (intrinsic mechanism)
• Vitamin K
  • Vitamin K is a co-factor to VKORc1 (vitamin K epoxide reductase complex subunit 1)
  • Post-translational modification of Factors II, VII, IX, X; proteins C and S
  • Converts 1st 7-12 glutamic acids to γ-carboxyglutamic acid
  • Confers calcium binding and lipid binding on these proteins
  • Without vitamin K, secrete des-γ-carboxyglutamic acid containing proteins (inactive in coagulation)

Hemostasis Summary

• Intrinsic: 12, 11, 9, 8
• Extrinsic: 7, 3
• Common: 10, 5, 2, Fibrinogen to Fibrin, 1

Fibrinolysis: Clot Dissolution

• Plasmin is formed from plasminogen - enzyme called activator (e.g. enzymes from urine (urokinase), tears, saliva or bacterial enzyme (streptokinase))
• Plasmin as an enzyme is involved in breaking down fibrin into soluble fragments (fibrinolysis)
• Plasminogen Plasmin
• Activator (e.g. t-PA, Kallikrein)
• Fibrin soluble fragments D-dimer
• t-PA: tissue plasminogen activator

Fibrinolysis: t-PA

• t-PA: tissue plasminogen activator
• Released from endothelial cells in response to vascular occlusion
• Protease that binds to N-terminus of fibrin via lysine residues
• Rapidly cleared from blood or inhibited by plasminogen activator inhibitor -1 and -2

Anticoagulants

• Intravascular clotting does not usually occur because:
  • Very small amounts of procoagulants are released
  • Presence of natural anticoagulants
• Antithrombin III : inhibits factor X and thrombin
  • Heparin from basophils and mast cells potentiates effects of antithrombin III (together they inhibit IX, X, XI, XII and thrombin)
• Antithromboplastin (inhibits tissue factors – tissue thromboplastins)
• Protein C and S: activated by thrombin; degrade factor Va and VIIIa

Abnormalities of Hemostasis and Thrombosis

• Thrombocytopenia (Platelets < 50,000/mcL; Normal 150-400,000/mcL)
  • Reduction in platelets count, leads to spontaneous bleeding
  • Results from:
    • Decreased production (radiation, cancer, infections, pollutants…)
    • Increased destruction (autoimmune diseases, heparin-induced)
    • Increased platelets consumption (Infections, cancer, pregnancy…)
• Disseminated intravascular coagulation (DIC)
  • Increased platelets consumption (Infections, cancer, pregnancy…)
• Vitamin K deficiency
• Hepatic failure
  • Clotting factors made by the liver
• Hemophilia A (VIII deficiency, most frequent) and B (IX deficiency): X-linked diseases, only males
• Von Willebrand’s disease (Loss of VIII constituents)

Tests of Coagulation

• ACT: Activated coagulation time
• PT: Prothrombin time
• INR: International normalized ratio
• PTT: Partial thromboplastin time
• APTT: Activated Partial thromboplastin time
• TT: Thrombin time
• Whole blood clotting time: Ear or finger
• D-dimer: Fibrin degradation products

Prothrombin Time (PT) Test

• Norm 11 -15 sec
• Evaluates extrinsic system (VII, X, V, II, fibrinogen)
• Prolonged PT indicates a deficiency in any of factors VII, X, V, prothrombin (factor II), or fibrinogen (factor I).
• Prolonged PT:
  • Vitamin K deficiency (vitamin K is a co-factor in the synthesis of functional factors II (prothrombin), VII, IX, and X)
  • Liver disease
  • Warfarin therapy
  • DIC (Disseminated intravascular coagulation)
  • Excessive heparin

International Normalized Ratio (INR)

• The result for the PT is expressed as a ratio (prothrombin clotting time for patient plasma divided by time for control plasma)
• $\text{INR} = (\frac{\text{patient PT}}{\text{Mean normal PT}})^{ISI}$
• Correction factor (ISI = International Sensitivity Index) is applied to the prothrombin ratio and the result issued as INR
• Target : INR~2-4 (depends on disease type)
• Application: Monitoring oral anticoagulant therapy (Warfarin)
• Notes: Heparin does not prolong INR (heparinase is included within the INR reagent), therefore, for heparin therapy aPTT and/or aPTT ratio are monitored

Activated Partial Thromboplastin Time Test (aPTT)

• Norm: 25-35 s
• Evaluates intrinsic system (VIII, IX, XI, XII, X, V, II, fibrinogen)
• An isolated prolongation of the aPTT (PT normal) suggests deficiency of factor VIII, IX, XI, or XII
• Prolongation of both the APTT and PT suggests factor X, V, II, or I (fibrinogen) deficiency, all of which are rare
• aPTT is normal in factor VII deficiency (PT prolonged) and factor XIII deficiency
• Most common case of prolonged aPTT – heparin!!!

Thrombin Time (TT)

• Norm: 14-15 sec
• Prolonged TT:
  • Heparin (much more sensitive to heparin than aPTT)
  • Hypofibrinogenemia

D-Dimer

• Norm: ≤ 500 ng/mL
• High levels:
  • Disseminated intravascular coagulation
  • Pulmonary embolism
  • Deep vein thrombosis
  • Sometimes: pregnancy, liver disease, cancer…

Abnormal Bleeding Time

• Prolonged bleeding time may indicate:
  • A vascular defect
  • A platelet function defect
  • Platelets count defect
• Drugs that may increase coagulation times include dextran, indomethacin, and salicylates (aspirin)
• Anticoagulant drugs

Selected Causes of Abnormal Coagulation Tests

Summary of Hemostasis

• Vascular injury or trauma, sub-endothelial proteins, such as Von Willebrand factor and collagen, are exposed and platelet adhesion occurs with the objective of promoting tissue healing
• The interaction between these proteins and platelets is mediated by a number of receptors at the platelet surface, and platelet activation occurs concomitantly to adhesion
• Multiple metabolic pathways are stimulated, leading to an increase in calcium intracellular concentration. This increase activates phospholipase A2 and actin-myosin ATPase, leading to thromboxane A2 formation and platelet conformational change, respectively
• Activated platelets release their granules (containing ADP, ATP, serotonin, calcium, fibrinogen, cytokines, and pro-thrombotic factors), increase their volume and reactivity
• Platelets aggregate through the binding of fibrinogen and glycoprotein IIb/IIIa receptor
• Fibrinolysis is the process to digest fibrin by the protease plasmin, that circulates naturally under an inactive form (plasminogen)
• Plasminogen is activated by tPA following tissue injury to limit over-coagulation