L-14 Hemostasis II

• Lecture covers Hemostasis II, a part of the Hematopoietic and Lymphoreticular System course (COM 5855) at Nova Southeastern University's Kiran C. Patel College of Osteopathic Medicine.

Learning Objectives

• Describe fibrinogen and its conversion to fibrin by thrombin (Factor II).
• Describe Factor XIIIa, a transamidase fibrin stabilizing factor.
• Describe the Common Pathway or final steps in the coagulation cascade.
• Describe the vitamin K dependent serine proteases Factors Xa and IIa.

Prevention of Inappropriate Primary Hemostasis

• Endothelial cells secrete nitric oxide (NO), a vasodilator, and prostacyclin (PGI2), which inhibits platelet aggregation.
• Heparin Sulfate activates anti-thrombin 3 (AT III), an anti-coagulant.
• AT III inactivates factors II, IX, and X.
• Thrombomodulin binds to thrombin (Factor II), which activates Protein C, which in turn inhibits Factors V and VIII.

Secondary Hemostasis: Intrinsic Pathway

• Called intrinsic because the factors needed for clotting are present within the blood.
• Triggered by negatively charged surfaces such as activated platelets, collagen, or glass.
• The liver produces coagulation factors (zymogens), and Factor XII is the first to join the intrinsic cascade.
• Damaged cells produce von Willebrand's factor (vWF), which binds to platelets and promotes platelet plug formation.
• Factor XIIa activates Factor XI to XIa.
• Factor XIa activates Factor IX to IXa.
• Factor IXa interacts with Factor VIII and Platelet Factor 3 (PF3), requiring Ca2+ for complex formation.
• The complex activates Factor X to Xa.

Thrombin Converts Fibrinogen to Fibrin

• Factor Xa cleaves Factor V into Factor Va, which interacts with Va and Ca2+ to form the prothrombin activator complex.
• Factor II (prothrombin) gets activated into thrombin (Factor IIa).
• Thrombin (Factor IIa) activates fibrinogen into fibrin.
• Factor XIII (fibrin-stabilizing factor) cross-links fibrin strands to create a stronger mesh.

Secondary Hemostasis: Extrinsic Pathway

• Called extrinsic because it requires tissue factor located outside the blood.
• Shorter and faster than the intrinsic pathway, it can form a clot in approximately 15 seconds.
• Triggered by the exposure of blood to tissue factor (TF) found in damaged endothelium.
• TF is also known as Factor III.
• Factor VII is exposed to TF and gets activated into Factor VIIa.
• Factor VIIa binds to TF and Ca2+, forming a VIIa-TF-Ca2+ complex.
• The VIIa-TF-Ca2+ complex cleaves Factor X into Factor Xa.

Thrombin Converts Fibrinogen to Fibrin (cont.)

• Prothrombinase complex cleaves prothrombin (Factor II) into thrombin (Factor IIa).
• Thrombin activates platelets and cleaves fibrinogen (Factor I) into fibrin (Factor Ia), which is insoluble.
• Thrombin activates cofactors V, VIII, and IX.
• Factor XIII is cleaved into XIIIa, which binds Ca2+ and cross-links fibrin chains to reinforce the fibrin mesh.
• Fibrin turns liquid plasma into a gel and traps formed elements.

Fibrin Cross-Linking Actions of Factor XIIIa

• Formation of stable fibrin clot that withstands mechanical and enzymatic breakdown.
• Patients deficient in FXIII form an initial clot, but it dissolves, leading to hemorrhage.

Common Pathway of Coagulation Cascade

• Once Factor X is activated to Xa, the pathways merge.
• Factor X is also called Stuart-Prower factor.
• Factor Xa joins Factor Va (plus Ca2+) to form the Prothrombinase complex.
• The Prothrombinase complex activates thousands of thrombin (F II) molecules.
• Thrombin can activate platelets and cleave fibrinogen.
• Factor XIIIa is important to stabilize the mesh.
• Factors II, VII, VIII, IX, X, XI, and XIII need Ca2+ cofactor in order to function.

Activation of Factor X

• Factor X is activated by the Intrinsic pathway, specifically the Tenase Complex (VIIIa-IXa-Ca2+).
• Factor X is also activated by the Extrinsic pathway, specifically the (TF-VIIa-Ca2+) complex.
• Both pathways lead to the formation of the Xa-Va-Ca2+ prothrombinase complex.
• This complex cleaves prothrombin (FII) to generate thrombin (FIIa).
• Amplification occurs as thrombin activates proteins of the intrinsic and common pathways.
• Thrombin also promotes the release of vWF and platelet activation, creating a "positive feedback" loop.

Overview of Clot Formation

• Intrinsic Pathway: Activated by factors present in blood, starting with Factor XII (Hageman factor), measuring aPTT (25-40 seconds).
• Extrinsic Pathway: Activated by muscle cell damage, starting with tissue factor (TF) and Factor VII, measuring PT (9.5-13 seconds).
• Common Pathway: Involves both intrinsic and extrinsic pathway components to form a cross-linked fibrin clot.

Clot Formation Regulation

• Anticoagulation: regulates clot formation, prevents clots from becoming too large which happens during primary and secondary hemostasis.
• Prevents formation of embolus.
• This is most important after production of thrombin.

Clot Retraction and Fibrinolysis

• Clot Retraction and Fibrinolysis: Happens after primary and secondary hemostasis.
• Helps the clot to contract and degrade.

Calcium and Coagulation

• Ca2+ is vital for coagulation.
• Component of: TF-FVIIa- Ca2+ complex, tenase complex, FVIIIa-FIXa- Ca2+, Prothrombinase complex FXa-FVa- Ca2+.
• Individual factors II, VII, IX and X (vitamin K-dependent factors) need Ca2+ as a cofactor for their activation.
• Coagulation is facilitated by platelet granular release of Ca2+.
• Coagulation is markedly impaired in hypocalcemia.

Blood Clotting Factors

• Factors I, II, III, IV, V, VII, VIII, IX, X, XI, XII, XIII play important roles in coagulation.
• Vitamin K is required for synthesis of factor II.

Vitamin K-Dependent Serine Proteases Factors Xa and IIa

• Vitamin K is a fat-soluble vitamin important for blood clot formation as well as bone health.
• It must be ingested and metabolized to create mature coagulation factors.
• It promotes the production of Factors II, VII, IX, and X.
• Deficiency in vitamin K leads to uncontrolled bleeding.
• Vitamin K hydroquinone acts as a cofactor to gamma-glutamyl carboxylase which converts Factors II, VII, IX and X into their functional forms.

Role of Vitamin K

• Catalysis by the enzyme glutamate carboxylase using oxygen, carbon dioxide, water, and protons produces a modified form of glutamate known as y-carboxyglutamate.
• Vitamin K is a cofactor for the glutamate carboxylase.
• y-Carboxyglutamate has a extra carboxyl group that is critical for the function of this protein because it allows the molecule to bind calcium.

Recycling of Vitamin K

• The carboxylation itself oxidizes (inactivates) vitamin K.
• The inactive form is called vitamin K Epoxide.
• Vitamin K epoxide reductase reduces vitamin K epoxide back to vitamin K Quinone, which regenerates active Vitamin K.
• Blood thinners can inhibit vitamin K epoxide reductase.
• Warfarin or coumadin are examples of blood thinners that inhibit vitamin K epoxide reductase.

Vitamin K Deficiencies

• The non-carboxylated factors are unable to bind to activating Ca2+ ions or to phospholipid membranes.
• Vitamin K deficiencies lead to disordered coagulation due to lack of function of Factors II, VII, IX, and X.
• Blocking both intrinsic and extrinsic pathways.
• Suspectible when one has Malabsorption (cystic fibrosis, celiac disease), Broad-spectrum antibiotics (fluroquinolones, cephalosporins), Premature neonates (breast milk is low in vitamin K).

Anti-coagulates

• Medications include Heparins, Vitamin K Antagonists, Direct Oral Anticoagulants (DOACs).

Hemostasis Drug Therapies

• Include Antiplatelet, Anticoagulants, Thrombolytics and Herbal Supplements

Proteins C and S

• Protein C and S work together to prevent your blood from clotting too much. Not enough could cause more clotting than you need.
• Protein C is a serine protease that destroys the activated cofactors Va and VIIIa and therefore inactivates them.
• It is activated when thrombin binds to thrombomodulin (at the surface of endothelial cells)
• Protein S is a cofactor that is needed by Protein C.
• Both are also vitamin K -dependent.

Deficiencies of Proteins C or S

• Not enough protein C or S could cause more clotting than you need.
• Cause thrombosis.
• Determined from blood sample.
• Protein C deficiency ~ 1:200-500 individuals
• Protein S deficiency ~1:500 individuals
• Treatment with warfarin decreases protein C activity