Secondary Hemostasis and Coagulation

Secondary Hemostasis

• Involves plasma coagulation
• Functions as a fibrin-forming coagulation system
• Produces a stable fibrin clot, strengthening the platelet plug
• Follows cascading reactions involving coagulation factors
• Generates thrombin to convert fibrinogen to fibrin

Coagulation

• Process where blood forms clots
• Occurs when plasma proteins, tissue factors, and calcium interact on platelets after vessel injury to form a fibrin clot
• Plays a role in the immune system

Coagulation Factor Nomenclature

• Fibrinogen: Factor I
• Prothrombin: Factor II
• Tissue Factor: Factor III
• Calcium: Factor IV
• Proaccelerin: Factor V
• Factor VI does not exist
• Proconvertin: Factor VII
• Antihemophilic Factor (AHF): Factor VIII
• Plasma Thromboplastin Component (PTC): Factor IX
• Stuart-Prower Factor: Factor X
• Plasma Thromboplastin Antecedent: Factor XI
• Hageman Factor: Factor XII
• Fibrin Stabilizing Factor: Factor XIII
• Prekallikrein: Fletcher Factor
• High-Molecular Weight Kininogen (HMWK): Fitzgerald Factor

Coagulation Factors

• Numbered by discovery date
• Each factor has one or more names
• Nomenclature is standardized by the International Committee on Nomenclature of Blood Clotting Factors (1958)
• Roman numerals are used

Characteristics of Procoagulants

• Fibrinogen is the only substrate that doesn't become an activated enzyme during the cascade
• Fibrinogen is the primary substrate for making fibrin; thrombin prefers fibrinogen
• Factors, Minimum Hemostatic Level (%), Mean Plasma Concentration (mg/dL), and In-Vivo Half-Life (hours)
• Factor I: 100%, 200-400 mg/dL, 100-150 hours
• Factor II: 20-40%, 10 mg/dL, 60 hours
• Factor V: 10-25%, 1 mg/dL, 24 hours
• Factor VII: 5-10%, 0.05 mg/dL, 6 hours
• Factor VIII: 25-30%, 0.01 mg/dL, 12 hours
• Factor IX: 15-25%, 0.3 mg/dL, 24 hours
• Factor X: 10-20%, 1 mg/dL, 48-52 hours
• Factor XI: 10-20%, 0.5 mg/dL, 48-84 hours
• Factor XII: 0-5%, 3 mg/dL, 48-70 hours
• Factor XIII: 2-3%, 2 mg/dL, 150 hours
• Prekallikrein: 35-50 ug/L, 35 hours
• HMWK: 5 ug/L, 156 hours

Kinin System

• Includes Prekallikrein, Kallikrein, Kininogen (LMWK and HMWK), and Kinins (bradykinins)
• Activated by coagulation and fibrinolytic systems
• The action of the system increases vascular permeability, vasodilation, hypotension, and contraction of smooth muscle

Coagulation Factor Classification by Function

• Zymogens are inactive forms circulating in the blood, activated only when needed
• Serine proteases cleave peptide bonds and include Factors IIa, VIIa, IXa, Xa, XIa, XIIa, and kallikrein
• Transamidases transfer amide groups; Factor XIIIa is a transamidase
• Activated coagulation factors denoted with the letter "a"
• Cofactors indirectly help activate zymogens and accelerate enzymatic reactions
• Cofactors: Factor III, Factor V, Factor VIII and HMWK
• Substrates are materials acted upon by enzymes

Contact Group

• Includes Factors XII, XI, HMWK, and Prekallikrein
• Involved in initial clotting phases
• Not consumed during activation
• Calcium- and Vitamin K-independent
• Activated by contact with negatively charged surfaces
• Deficiencies usually asymptomatic, except for Factor XI deficiency, which results in mild bleeding disorders

Prothrombin Group

• Adsorbable, affected by Coumadin, not consumed during clotting, and calcium- and Vitamin K-dependent
• Activated Prothrombin forms the main coagulation enzyme

Factor VII

• Stable factor with a short half-life of 4-6 hours
• Production halts; and it rapidly disappears from the blood

Factor IX

• Known as Christmas factor, named after Stephen Christmas, the first patient to have Christmas disease (Hemophilia B) in 1952

Factor X

• Also known as Stuart-Prower factor; deficiency identified in the late 1950s in patients named Stuart and Prower

Tissue Factor (III)

• A lipoprotein that initiates the extrinsic pathway
• Doesn't circulate unless damage to tissues occurs
• Found in all cells except those in blood
• High concentrations in lung, brain, placenta, heart, kidney, and testes
• Leukocytes express this during inflammation, initiating coagulation

Fibrinogen Group

• Thrombin-sensitive
• Found in platelet alpha granules (I, V, VIIIR:Ag)
• Consumed during the process
• Calcium-dependent, vitamin K-independent, and an acute phase reactant

Fibrinogen

• Most abundant coagulation factor
• Forms the adhesive protein that forms the fibrin clot and acts as an APR (Acute Phase Reactant)

Factor V

• Labile, unstable and deteriorates quickly and most unstable of the coagulation factors

Factor XIII

• Transamidase that cross-links fibrin clots

Factor VIII

• Circulates as VIII/vWF (vWF >90% of complex) Composed of two fractions:
  • Factor VIII:C: the coagulant portion, measured by standard assays
  • Factor VIII:Ag: the antigenic portion, measured by immunoassays

Calcium & Vitamin K

• γ-carboxyglutamic acid: critical for calcium binding
• Vitamin K is required for γ-carboxylation of factors 2, 7, 9, and 10
• Allow vitamin K-dependent factors to bind Ca ++ and form calcium bridges with phospholipid on platelet surface

Coagulation Cascade

• Includes serial activation of inactive plasma proteins, phospholipids, and calcium to transform circulating substances into an insoluble gel
• Each coagulation factor is converted to an active form by the preceding factor in the cascade
• Calcium serves as a cofactor in some reactions
• Blood coagulation occurs on cell surface membranes Subendothelial tissue exposed with vascular injury and platelet surface provide the critical membranes Clotting factors bind to the phospholipid membrane surface

Coagulation Pathways

• The pathways for the formation of these complexes include
  • intrinsic, extrinsic, and common pathways
• Both intrinsic and extrinsic converge to share factors on the common pathway

Extrinsic Pathway

• Starts with tissue factor and calcium activating factor 7 to 7a
• Factor 7a activates factor 10 to 10a
• Factor 7a activates factor 9 in the intrinsic pathway to 9a

Common Pathway

• Both intrinsic and extrinsic pathways converge
• Both pathways activate Factor 10 to 10a
• Step 1: 10a in the presence of Factor 5, Ca++, phospholipid converts prothrombin to thrombin
• Step 2: Thrombin feeds back to activate factors 8 and 5
• Step 3: Thrombin converts fibrinogen to soluble fibrin
• Step 4: Thrombin converts factor 13 to 13a
• Step 5: 13a cross-links the fibrin monomers to form a stable fibrin polymer

Intrinsic Pathway

• Clot formation factors are in the vascular compartment
• Prekallikrein, HMWK,Factors XII, XI, X, and VIII are involved
• Initiated by negatively charged foreign substances

Intrinsic Pathway Steps

1. Factor XII activates to XIIa
2. XIIa interacts with Prekallikrein and Kininogen to activate more Factor XII to XIIa
3. XIIa activates factor XI to XIa
4. XIa activates Factor IX to IXa in the presence of Ca++
5. IXa participates with co-factor VIII:C, Ca++, and PF3, to activate Factor X
   • IXa– factor VIIIa– phospholipid – Ca++ = tenase complex

Extrinsic Pathway Steps

• Dominant in vivo pathway
• Starts when there is injury to a blood vessel wall
• Initiates when a tissue factor not found in blood enters the vascular system

Initiation and Propagation of Thrombin

• VIla binds to tissue factor (TF) and activates both factors X and IX
• Cell-bound factor Xa combines with Va and generates a small amount of thrombin, activating platelets, V, VIII, and XI and begins fibrin formation
• Factor XIa combines with factor VIIIa on the platelet surface to activate X, which forms prothrombinase (Xa:Va)

Conversion of Fibrinogen to Fibrin

• Fibrinogen alpha and beta fibrinopeptides are cleaved by thrombin, forming soluble fibrin monomersFibrin monomers spontaneously polymerize, forming soluble fibrin polymers.

Thrombin Feedback

• Positive: Low thrombin levels activating factors 5, 8 & 13 and platelet activation
• Negative: high thrombin binds to thrombomodulin which activates protein C & plasminogen coverts to plasmin

Fibrinolysis

• Dissolution of fibrin clots
• Final stage of coagulation that defends against occlusions of blood vessels
• Regulated by activators and inhibitors, mainly PLASMIN

Physiologic Blood Coagulation

• “Contact phase” factors (F12, HK, and PK) are not believed to contribute to normal physiologic hemostasis
• Under normal conditions, F7a alone does not contribute to clot formation in significant way.
• Natural clotting is started as soon as enough TF/F7a exists to trigger activation of F9 to F9a

Plasminogen

• Glycoprotein which is a zymogen produced in the liver
• Activated form is "plasmin:" converted to by “plasminogen activators”

Plasminogen Activators

• Intrinsic: Factor XIIa, Kallikrein, HMWK
• Extrinsic: Tissue-type plasminogen activator (t-PA) created by veins & Urokinase-like (u-KA) created by kidney
• Therapeutic/Exogenous: Streptokinase, Urokinase, TPA are used to treat thromboembolism

Plasmin

• Degrades fibrin clots, fibrinogen, Factor V& VIII
• Not normally found during circulation

Fibrinolysis Mechanism

1. Plasminogen bound into the fibrin clot is formed
2. Plasminogen activator (t-PA) is obtained from surrounding endothelial cells
3. The t-PA absorbs to activate plasminogen
4. Plasmin dissolves Non-crosslinked fibrinogen > Fragment X,Y, D Crosslinked fibrin > Fragment YY/DXD, YD/DY & DD/E

D-Dimer (fragment DD)

• FDP is produced thru fibrinolytic action & indicates that its been lysed
• Increased D-dimer: Pulmonary embolism, Deep vein thrombosis, DIC & Sickle cell disease

Inhibitors of Fibrinolysis

• Inhibitors to "plasminogen activators:" inhibitor-1 (PAI-1), made by endothelial, platelets & Inhibitor-2 (PAI-2)
• Inhibitors to “activation of fibrin bound plasminogen:” thrombospondin
• Inhibitors to plasmin include α2-Antiplasmin which is the primary inhibitor, α2-Macroglobulin & α1-Antitrypsin

Thrombin-Activatable Fibrinolysis Inhibitor (TAFI)

• Plasma procarboxypeptidase synthesized in liver, becomes activated the thrombin
• TAFI inhibits Fibrinolysis by removing binding sites

Inhibitors of Coagulation

• Protein C
• Protein S
• Antithrombin III
• TFPI

Protein C

• Degrades factors Va & VIIIa
• Stimulates fibrinolysis by deactivating plasminogen activation

Protein S

• Protein is in cell membranes as a receptor
• Protein binds is used to inactivate factors Va and VIIIa
• 40% plasma is free, 60% is bound to C4BP

Antithrombin III

• Principal inhibitor of coagulation
• Inhibits hemostatic enzymes; IIa, VIIa, IXa, Xa, XIa, XIIa and kallikrein
• Factors are reduced thru factors - IIa & Xa

Tissue Factor Pathway Inhibitor (TFPI)

• Is the most potent inhibitor of the FVIIa–tissue factor complex
• Does not inhibit thrombin
• Cerebral anticoagulant