Assessing Coagulation
The Coagulation System

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• The Platelet
• The Revised Coagulation Pathway
• Regulation of the Coagulation Pathway
• The Fibrinolytic Pathway
• Regulation of the Fibrinolytic Pathway

The Platelet

1. Vascular injury allows reflex vasoconstriction with exposure of the subendothelial matrix and reduced local blood flow.
   
2. The starting point is the attachment of the soluble protein, von Willebrand factor, to the subendothelial matrix.
   Failure of this step may be due to
   1. Absence of von Willebrand factor - Autosomal Dominant and Recessive inheritance
   2. Malfunction of collagen - Scurvy

   Tests

   1. History and examination
   2. Ristocetin cofactor test for vWf
3. Platelet adhesion. Normal von Willebrand factor, when bound to the subendothelium, exposes multiple intrinsic binding sites for the platelet specific membrane Glycoprotein Ib (GPIb).
   Failure of this step may be due to
   1. Occupancy of the GPIb receptor. Dextran, multiple myeloma and idiopathic thrombocytopaenia purpura can occupy the GPIb receptor.
   2. The number of platelets - Results of the bleeding time become abnormal when the platelet count falls below 100x10⁹/L, however spontaneous small-vessel bleeding does not increase until the platelet count is less than 5x10⁹/L.
   3. Delivery of the platelets - Platelets are normally concentrated in the peripheral blood stream. A functional dilution of platelets within the column of blood occurs when the packed cell volume is less than 20%
   4. Absence of the GPIb molecule. Proteolytic degradation during Cardiopulmonary bypass and storage of greater than 3 days causes loss of GPIb. The Bernard-Soulier Syndrome is a congenital lack of the protein.
4. Platelet aggregation. The bound platelets disintegrate to initiate a platelet binding cascade.
   1. von Willebrand factor-GPIb binding stimulates platelet disintegration which exposes the Glycoprotein IIbIIIa binding site for further attachment of von Willebrand factor and fibrinogen.
   2. Dense granules in the platelets release ADP, enhancing platelet aggregation and disintegration, serotonin and thromboxane A₂ which cause vasoconstriction and Calcium. Calcium is essential in the activation of the soluble proteins of the coagulation pathway.
   3. Alpha granules from the platelets release the coagulation proteins - fibrinogen, thrombospondin, fibronectin, factor V and factor VIII
   4. The phospholipid phosphatidylserine (Platelet Factor 3) is exposed on the surface of the disintegrating platelet, serving as an essential base for the initiation of soluble proteins in the coagulation pathway

   
   Failure of this step

   1. Insufficient number of platelets.
   2. Dysfunctional platelets.
      1. Prior activation occurs during cardiopulmonary bypass, storage, exposure to aspirin, uraemia and acute and chronic alcohol exposure.
      2. Congenitally impaired function - absence of Gp IIb-IIIa ("Glanzmann's thrombasthenia")

   
   Tests of the above axis

   1. History and examination
   2. Platelet count
   3. Platelet aggregometry
   4. Red blood cell count
   5. A suggested practical test of the integrity of the entire axis is a correctly performed
      bleeding time.

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The Revised Coagulation Pathway
				Tissue Injury
XIIa??
XI XIa		IX IXa			X Xa
					Thrombin		prothrombin
			XIII
cross-linked fibrin				fibrin	fibrinogen

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Key:

Stimulates:

Inhibits:

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1. There is a rapid initiation of coagulation when "Tissue Factor" (a protein-phospholipid complex normally present on vascular cells and activated monocytes), is exposed to factor VII in the presence of calcium.
2. The activated Tissue factor-VII complex activates factors IX and X
   1. Factor IXa enhances the production of Xa, especially in the presence of the co-enzyme VIIIa.
   2. Factor Xa converts Prothrombin to Thrombin (factor IIa). This is greatly facilitated by the presence of the coenzyme Va.
   3. Thrombin enhances its own generation by
      1. Activating the coenzymes V and VIII
      2. Activating factor XI, this generates more IXa and more XIa by positive feedback.
      3. Encouraging platelet aggregation and disintegration
3. Thrombin cleaves fibrinogen yielding monomers of fibrin which then polymerises to form the fibrin clot. Factor XIII, activated by thrombin and Ca²⁺, stabilises this clot by forming covalent bonds between the fibrin molecules.

Thrombin's Positive Feedback!
				Tissue Injury
XIIa??
XI XIa		IX IXa			X Xa
						Thrombin

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Failure of normal coagulation

1. Inadequate activity of a factor - A 30% of normal activity is clinically significant. The actual concentration of the proteins in the blood varies considerably and is not important by itself.
   1. Factor VIII - Classical Haemophilia A
      1. Factor VIII deficiency
      2. von Willebrand factor deficiency
   2. Factor IX deficient - "Christmas Disease" Haemophilia B
   3. Factor XI deficient
   4. Factor V deficient
   5. Factor X or II deficient
   6. Factor XIII deficient
   7. Vit K dependant serine proteinases (II, VII, IX and X) deficient
      1. Coumarin anticoagulants
      2. Vit K deficiency
      3. Severe liver disease
2. Presence of an inhibitor
   1. Lupus anticoagulant directed against the phospholipid
   2. Factor V inhibitor
   3. Factor VIII inhibitor
   4. Factor IX inhibitor
3. Inappropriate regulation of the pathway

Tests of normal coagulation

1. History and Examination
2. Whole blood clotting time
3. Prothrombin Time
4. Activated Partial Thromboplastin Time
5. Screening Mixing Test
6. Russel viper venom test
7. Liver function tests
8. Thrombin Time
9. Lupus anticoagulant detection
10. Evaluation of regulation pathway

Regulation of Coagulation
				Tissue Injury
XIIa??
XI XIa		IX IXa			X Xa		EPI
				Antithrombin III, heparan sulphate
				Thrombin
				Proteins C,S

Inherent inhibitors of coagulation control this explosion of coagulation material

1. Intact endothelium secretes Prostaglandin I₂ which prevents platelet activation and causes vasodilatation
2. Antithrombin III, a circulating serine protease inhibitor (serpin) and heparan sulphate from the endothelium inactivate thrombin, IXa, Xa and XIa.
3. Protein C, activated by the thrombin-thrombomodulin complex, inactivates Va and VIIIa in the presence of the activated cofactor Protein S
4. Extrinsic Pathway Inhibitor is a double headed protease inhibitor. One head binds to Xa and the second binds to the Tissue Factor-VIIa. This inhibition is overcome when there is a large amount of Tissue Factor present.
5. Damaged endothelial cells release tissue plasminogen activator, stimulating the fibrinolytic pathway

Failure of this pathway

1. Antithrombin III deficiency
2. Protein C deficiency
3. Protein S deficiency
4. Inherited resistance of Va to cleavage by activated Protein C

Tests

1. Specific protein assays
2. Factor V susceptibility test

Normal Fibrinolysis
		Tissue Injury
XII		XIIa	Release of..          tPA, urokinase
	prekallikrein	Kallikrein
		plasminogen		Plasmin
			fibrin		fibrin degradation products, D-Dimer
			Va, VIIIa, GPIb		Inactivated

Fibrinolysis is initiated at the time of tissue injury

1. Tissue plasminogen activator and Urokinase are released from endothelial cells following injury and in response to thrombin. They cleave plasmin from plasminogen bound to fibrin within the clot
   1. Plasmin degrades fibrin into D-Dimers and Fibrin Degradation Products.
   2. Plasmin degrades Va, VIIIa and GPIb
2. The negatively charged subendothelium activates factor XII, which then releases kallikrein from prekallikrein, especially in the presence of high molecular weight kininogen.
   1. Kallikrein activates plasmin from plasminogen
   2. Kallikrein releases Bradykinin from kininogen, setting up the pro-inflammatory process.

Regulation of Fibrinolysis
		Tissue Injury
XII		XIIa
			tPA, urokinase
	prekallikrein	Kallikrein
		Plasminogen Activator Inhibitor 1, (tPA & UK also inhibited by alpha2 antiplasmin, alpha 2 macroglobulin)
		plasminogen		Plasmin
					Alpha-2 antiplasmin (forms PAP complexes)

The control pathway is set in motion during platelet disintegration by the release of Plasminogen Activator Inhibitor 1. Inherent inhibitors to the fibrinolytic pathway also exist.

1. Plasminogen Activator Inhibitor 1
   1. Prevents tissue plasminogen activator and urokinase from activating circulating plasminogen to plasmin, and the activation of prekallikrein to kallikrein
   2. Is inhibited by binding with activated protein C
2. Alpha 2 Antiplasmin and alpha 2 macroglobulin
   1. Prevent the formation of plasmin from circulating plasminogen by tissue plasmin activator and urokinase
   2. Bind and inactivate free plasmin, creating a plasmin-alpha2 Antiplasmin (PAP) complex.

Failure of this pathway

1. Deficient Plasminogen Activator Inhibitor 1 or 2
2. Deficient alpha 2 Antiplasmin - Autosomal Recessive
3. Presence of extrinsic plasmin inhibitors - Epsilon aminocaproic acid, tranexamic acid
4. The combination of streptokinase and plasmin is not inactivated by alpha 2 antiplasmin

Tests

1. Specific alpha 2 antiplasmin assay

Web page author: lara@anaesthetist.com