Secondary Hemostasis & Coagulation Factors

Definition of Terms

• Plasma Factor: A coagulation factor found in plasma.
• Platelet Factor: A coagulation factor found in platelets.
• Zymogen: Refers to an inactive substance that is converted into an enzyme when activated by another enzyme.
• Serine Protease: An active enzyme, capable of hydrolyzing arginine or lysine-containing peptide bonds.
• Small "a": Indicates an activated coagulation factor.
• Cascade: Refers to a series of activations.
• Fibrin Clot: Represents the final product or end stage of coagulation.
• Cofactor: Assists in accelerating the zymogen activation process.

Secondary Hemostasis

• Goal of secondary hemostasis is to create a stable fibrin clot.
• Two pathways include the extrinsic and intrinsic pathways.
• The series of reactions involves coagulation factors
• The four components are:
• Enzyme precursors (zymogens) which include factors II, VII, IX, X, XI, XII, and Prekallikrein.
• Non-enzymatic cofactors consist of factors V, VIII, HMWK, and Tissue Factor.
• Calcium.
• Phospholipid.

Role of Coagulation in Hemostasis

• Upon vessel injury, plasma proteins, tissue factors, and calcium interact to form a fibrin clot
• The International Committee on Nomenclature of Blood Coagulation Factors assigns Roman numerals to plasma factors.
• Calcium and Tissue Thromboplastin are the 2 exceptions to the above point, listed as IV and III respectively.
• Calcium and Tissue Thromboplastin normally circulate in plasma as inactive proteins.
• For the majority of plasma factors, activation occurs in a cascade-like manner.
• Zymogens must first be converted into an enzyme known as serine protease, which then activate other factors.

Coagulation Factors

• Liver is the primary production site, except for factors III and IV.
• Factor VIII is produced by other tissues in addition to the liver.
• vWF:VIII is the product of megakaryocytes and endothelial cells.
• Liver disease can cause an increase in factors I and VIII, as a compensation mechanism.
• Factor VII possesses the shortest half-life at 6 hours.
• Factor VII being an extrinsic pathway factor, is reduced early in Acute Liver Dysfunction.
• Prothrombin time is a good test to assess liver function.
• All coagulation factor diseases are AUTOSOMAL RECESSIVE, with the exception of Factor VIII and IX deficiency, which are X-LINKED RECESSIVE.

Factor I (Fibrinogen)

• Normal concentration is 200-400mg/dL
• This is the most concentrated of all plasma procoagulants.
• Involved in platelet aggregation and is derived from alpha granules
• Levels increase by ~10mg/dL per decade in elderly individuals.
• Levels of concentration <100mg/dL result in prolonged PT and APTT.

Factor III (Tissue Factor/Thromboplastin)

• Normal concentration is 10mg/dL
• Not present in the plasma.
• Considered never deficient due to its production by injured tissues.

Factor IV (Calcium Ions)

• Normal concentration is 8-10mg/dL

Factor V (Proaccelerin)

• Normal concentration is 1mg/dL
• Deficiency in factor V known as Owren's Disease or Parahemophilia.
• Factor V Leiden :
• A mutated abnormal form of factor V.
• Not affected/inactivated by the ProteinC-Protein S complex, which leads to excessive clot formation and possible cause of thrombophilia.

Factor VII (Proconvertin/Stable Factor)

• Normal concentration is 0.05mg/dL
• First factor affected by Warfarin therapy.
• Has a short half-life of 6 hours.

Factor VIII (Antihemophilic Factor A)

• Concentration is 0.01mg/dL
• Unstable and must be bound with von Willebrand factor (vWF) in plasma.
• Hemophilia A (Classic Hemophilia) : Deficiency of Factor 8
• Reduced levels of the procoagulant portion (VIII:C) are decreased in Hemophilia A.
• Assays include standard Factor VIII assay and APTT
• Factor VIII:Ag is the antigenic portion
• Antigens are determined via Immunoassay

Von Willebrand factor (vWF)

• Normal concentration is 1mg/dL
• The largest molecule in human plasma.
• Receptors exist for platelet and collagen.
• Key platelet receptor, GPIb/IX/V is the primary receptor for vWF

IX (Christmas Factor/ Antihemophilic B)

• Normal concentration is 0.3mg/dL
• Deficiency leads to Hemophilia B, also called Christmas Disease.

Factor XI (Antihemophilic factor C)

• Normal concentration 0.5mg/dL
• Deficiency leads to Hemophilia C, also known as Rosenthal Syndrome.
• Results in bleeding
• It affects 50% of Ashkenazi Jews.

Factor XII (Hageman Factor/Glass Factor/Contact Factor)

• Deficiency causes a thrombotic tendency.
• Does not result in bleeding

Factor XIII (Fibrin Stabilizing Factor)

• Normal concentration of 2mg/dL.
• Not needed for clotting; helps to stabilize clot.
• Prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT) cannot detect Factor XIII deficiency. To check for deficiency:
• 5M Urea Clot Solubility Test (Duckert's Test)
• Soluble: Factor XIII is deficient.
• Insoluble: Factor XIII is present

Factor PK (Prekallikrein/ Fletcher Factor)

• Normal values are 35-50ug/mL

Factor HMWK

• Normal values are 5mg/dL
• (High Molecular Weight Kininogen)/ Reid Factor/ William's Factor/ Fitzgerald/ Flaujeac

Platelet Phospholipids

• Also known and collectively called as Platelet Factor 3.

Thrombin Feedback Mechanism

• The purpose is to control and balance the coagulation processes to prevent clotting and bleeding.
• Thrombin has two roles:
• Activator
• Inhibitor

Thrombin as an Activator

• Autocatalytic and self-perpetuating.
• Enhances prothrombinase complex effects.
• Activates factors V and VIII.
• If present in plasma, enhances FV and FVIII which stimulates aggregation.
• Acts as a trypsin-like enzyme, converting fibrinogen into fibrin monomers.
• Activates FXIII to stabilize fibrin resulting in a soluble clot

Thrombin as an Inhibitor

• At higher concentrations, it destroys V and VIII (unexplained reason)
• It activates Protein C, a potent anticoagulant.
• Activation enhanced by Calcium + thrombomodulin + Protein S cofactor to enhance Protein C
• Protein C-S complex fibrinolysis, increasing plasminogen activator activity and inhibiting t-PA inhibitor
• Thrombin + Thrombomodulin loses it Enhance V and VIII activity
• Stimulating platelet aggregation
• Converting I to Fibrin

Contact Factors

• Include HMWK, Prekallikrein, XII, and XI.
• They are absorbed by a negatively charged surface (collagen).

Prothrombin or Vitamin K-dependent factors

• Include Factors IX, X, VII, and II.
• Synthesized by liver in the presence of Vitamin K (cofactor).

Inhibitors of Vitamin K-dependent factors

• Dietary vitamin K deficiency
• Administration of antibiotics
• Oral anticoagulant therapy (coumarin warfarin)
• Result: Can't bind to platelet phospholipid

Fibrinogen Group

• Consist of I, V, VIII, XIII.
• The characteristics include:
• Highest molecular weight.
• Most labile.
• Consumed in coagulation.
• Only group that acts as substrates for plasmin.
• The only factors of fibrinogen group found in platelet (alpha-granules) are I and V.
• Factor XIII found in platelet cytoplasm.
• Factor VIII:C is the coagulant portion not found in platelets.

Coagulation Pathways

• The Extrinsic pathway: Factor VII, Tissue Factor III.
• The Intrinsic pathway: Factor XII, XI, IX, VIII.
• The Common Pathway (Factor V, X, II, I, XIII).

Extrinsic Pathway

• Activated by the release of Tissue Thromboplastin from injured tissue cells
  • IIIa activates VII to VIIa (serine protease)
  • VIIa (plus platelet phospholipid and Ca++) activates X to Xa
• Named as it necessitates adding Tissue Extract (PL) to plasma samples in vitro to study the extrinsic coagulation factors.

Intrinsic Pathway

• Activated by the contact of contact factors (XII, XI, HMWK) with collagen.
• Collagen exposure from broken endothelium activates XII to XIIa
• XIIa activates XI to XIa with Ca2+ which activates IX to IXa
• IXa activates X to Xa with Ca2+, platelet phospholipid and VIIIa
• XIIa then activates Prekallikrein w/ HMWK to Kallikrein
• Kallikrein (w/ HMWK) activates XII to XIIa
• Intrinsic name is derived from the fact that coagulation factors that participate in this pathway are found circulating in the plasma

Common Pathway

• Key factor activation of X to Xa by either the intrinsic or extrinsic pathway
  • Intrinsic or Extrinsic then activates X to Xa
  • Xa activates II to IIa (Thrombin)
  • IIa activates I to fibrin clot (SOFT and SOLUBLE)
• Finally, the Fibrin clot is then stabilized by XIII to make it HARD/STABLE

Process of Fibrin Clot Formation

• Theories include:

1. Morawitz (1905) two stage process
   • Key factors are: Prothrombin, Calcium, Thrombokinase, Thrombin, and Fibrinogen
2. Cascade or Waterfall (1964). Accepted model.
   • Extrinsic and Intrinsic pathway that differ in the activation of Factor X
   • Both pathways have zymogens which will become active enzymes
   • Interactions with platelet, fibrinolysis, complement and kinins occur.

Final Clot Formation

• Begins with Thrombin converting to Fibrin.
• Fibrinogen/Factor I is a key factor as a Glycoprotein with 3 different but interwoven chains:
• alpha – 16 amino acids
• beta- 14 amino acids
• And gamma
• Alpha and Beta at the terminal makes = 4 total fibrinopeptides

Conversion of Fibrinogen to Fibrin

• First step: Thrombin cleaves 4 fibrinopeptides (alpha, beta)
• Fibrinopeptides are removed resulting in soluble fibrin monomer (unstable gel)
• Second step: Fibrin monomers bind side-to-side and end-to-end through electrostatic bonds only to form an aggregate
• This results in fibrin polymers or strands, that still soluble
• However, it is soluble and can be dissolved IN VITRO by 5molar urea or Weak acids (1% monochloracetic acid) and Plasmin dissolution
• Third step: Clot stabilization
• Requirements: Thrombin, Calcium, FXIII
  • Thrombin (w/ calcium) activates FXIII to FXIIIa
  • Factor XIIIa (“transamidase” or “transglutaminase") crosslinks adjacent monomers through formation of covalent bonds
• Alpha and Gamma chains help in establishment of stable fibrin clot