Aniara | Shaping the Future with Innovative Solutions

Coagulation Corner

Tuesday, November 8, 2016

The Coagulation Cascade

Written By Donna Castellone, MS, MT (ASCP) SH | LinkedIn

Ah, the mysteries of life- the Bermuda triangle, the lost city of Atlantis, the WOW theory, and Mayan civilization. Some might even add the coagulation cascade to that list. It instills fear and confusion in students, residents and technologists alike. It is complicated with all factors, and inhibitors displaying the intrinsic, extrinsic, common and fibrinolytic systems. Or is can be as simple as- tissue factor becomes activated, then a miracle occurs and a clot forms! It was Paul Morawitz in 1905 that assembled coagulation factors into the scheme of coagulation which demonstrated that in the presence of calcium and thromboplastin, prothrombin (II) was converted to thrombin which in turn converted fibrinogen (I) into a fibrin clot. This theory persisted for 40 years until Paul Owren, in 1944, discovered a bleeding patient that a four factor concept of clotting could not apply, thus factor V was discovered.

The coagulation cascade is a series of enzymatic reactions in which you have unactivated factors (zymogens) that become activated (serine proteases) to form the final product of a fibrin clot. The entire process is kept in check with a series of inhibitors. Most of them are plasma proteins synthesized by the liver (vitamin K is needed for the synthesis of factor II, VII, IX and X).Several of these steps require Ca++ and platelet phospholipid.

The functional enzymes in the coagulation cascades are termed coagulation factors and are assigned a specific roman number and are called Zymogens or enzyme precursors (II, VII, IX, X, XI, XII, Prekallkrein). When activated become serine proteases. Cofactors are nonenzymatic (V, VIII, HMWK, Tissue factor(thromboplastin).

Factors are also placed into groups:

  • Fibrinogen group: I,V,VIII,XIII
    • Most labile, are consumed in coagulation, found on platelets
  • Prothrombin group: II,VII,IX,X
    • Vitamin K dependent, may be affected by coumarin,diet, antibiotics
  • Contact group: XI,XII,HMWK, Prekallikrein
    • Initiate intrinsic path and fibrinolysis
The in-vitro cascade allows logical effective lab based screening which can be evaluated through the PT & APTT. However, this doesn't reflect clotting physiologically, but it does play a role in laboratory evaluation of a potential bleeding or clotting disorder. It is divided into the extrinsic, intrinsic and common pathway.

Extrinsic pathway
Requires contact with tissue factors external to blood. This occurs when there is trauma to the vascular wall and surrounding tissues. The extrinsic system is triggered by the release of tissue factor (thromboplastin from damaged tissue) which binds to calcium and, that activates factor VII, forming the TF;VIIa complex. This in turn converts FX to FXa which now becomes the TF:VIIa +Ca +PF3 or the EXTRINSIC TENASE complex which then feeds into the common pathway.

Intrinsic Pathway
The initial reaction is the conversion of inactive factor XII to active factor XIIa. Factor XII is activated in vitro by exposing blood to foreign surface (glass test tube). Activation in vivo occurs when blood is exposed to negatively charged collagen fibers underlying the endothelium in the blood vessels.

Initially FXII is auto-activated to XIIa and binds to high molecular weight kininogen (HMWK) and forms a complex that binds to the subendothelial collagen layer. FXIIa in the presence of prekallikrein (PK) and HMWK converts FXI to FXIa. FXIa + Ca2+ converts FIX to FIXa [FIXa + FVIIIa + Ca2+ + PF3] complex or the "INTRINSIC TENASE complex. FVIIIa – a cofactor orients FIXa and FXa in the proper orientation to attach to the platelet phospholipid surface (PF3) The intrinsic tenase complex" converts FX to FXa which then feeds into the common pathway.

Common Pathway
In the common pathway [FXa + FVa + PF3 + Ca2+] - prothrombinase complex converts prothrombin (II) to thrombin (IIa) . FVa serves as a cofactor which positions the tenase complex (extrinsic or intrinsic) to interact with FII on the platelet phospholipid surface. Prothrombin is converted into thrombin (IIa) which then converts circulating fibrinogen into soluble fibrin monomers. Thrombin converts FXIII to FXIIIa which cross links the fibrin monomers into an insoluble fibrin polymer or a clot.

The clot then needs to be dissolved and that happens during fibrinolysis. The presence of fibrin triggers the activation of plasminogen to plasmin. Plasminogen is the target substrate of the activation systems and the main enzyme in the fibrinolytic system. After cleavage, plasminogen becomes the active enzyme plasmin. Plasmin then splits fibrin and fibrinogen into fragments. This interferes with thrombin activity, platelet function, and fibrin polymerization, leading to clot dissolution. These fragments are removed by the RES.

Activators and Inhibitors
Tissue plasminogen activator (t-PA) hydrolyzes plasminogen bound to the fibrin clot and initiates fibrinolysis. Plasminogen Activator Inhibitor (PAI-1) prevents t-PA and urokinase from activating free fluid-phase plasminogen. While α2-Antiplasmin rapidly inhibits free plasma.

Antithrombin (AT) - major regulator of coagulation cascade that inhibits factors IIa, Xa, and the other serine proteases. It also serves as a cofactor for heparin and other heparinoids. Heparin Cofactor II (HCII) is similar to AT, but only inhibits factor IIa.

Protein C when activated becomes Activated Protein C (aPC) which inhibits Va and VIIIa. While Protein S serves as a cofactor to PC. Tissue Factor Pathway Inhibitor (TFPI) - inhibits extrinsic tenase (TF:VIIa +Ca +PF3) while Thrombin Activatable Fibrinolysis Inhibitor (TAFI) TAFI is an enzyme that circulates in plasma and is involved in the regulation of fibrinolysis. TAFI when activated to TAFIa, functions to suppress fibrinolysis by the removal of lysine residues from the fibrin clot that are exposed as fibrin is degraded by plasmin. In this way tissue plasminogen activator (tpa) binding is restricted and further activation of plasminogen to plasmin, prevented and so the fibrin clot at the site of vascular injury is protected from fibrinolysis.

Cell Based Model of Coagulation
This is the process of physiological coagulation which accounts for all of the cellular components in coagulation. This occurs in 3 phases:

  1. Initiation
  2. Amplification
  3. Propagation

In this process VIIa forms as usual via binding of VII to TF and VIIa activates some X→Xa. FXa + Va converts prothrombin (FII) to thrombin (IIa) . This results in the initial burst of thrombin (IIa). Thrombin in turn feeds back up to activate FV, FVIII, FXI, FXIII and the TF:VIIa complex also converts FIXα to FIXa and FIXa + FVIIIa also converts FX to FXa. Xa converts a small amount of prothrombin to thrombin; this thrombin is used to produce small amounts of VIIIa and Va. As the concentration of TF-VIIa-Xa-thrombin increases, Tissue Factor Pathway Inhibitor inactivates this complex stopping further production of thrombin.

Initiation of coagulation occurs when sub-endothelial tissue is exposed to the circulation at a site of injury. These tissues express tissue factor at their surface, which binds to endogenous activated FVII. This complex binds small amounts of FX and FV to the exposed endothelial surface, which produce small quantities of thrombin. The thrombin activates platelets that are attracted to the site by the process, as well as other plasma-borne clotting factors. The activated factors (among them FVIII and FIX) enable the binding of activated FX and FV to the surface of platelets whose activation has produce conformational changes in their surface membranes to expose the 'reaction sites' necessary for continuation of the process. This leads to the 'thrombin burst' that is necessary for the large-scale production of fibrin and so the development of an effective clot.

What is the Role of Thrombin?
Thrombin (IIa) activates FV→Va as the prothrombin complex which assembles on platelet membranes. Thrombin releases VIII→VIIIa as a component of a tenase complex which assembles on platelet surfaces. Thrombin (IIa) activates XI→XIa and IX→IXa. IIa then leads to platelet activation to set the stage for tenase & prothrombin complex.

Step Two is Amplification:
In this phase, the small amount of FXa produced by TF-rFVIIa interaction leads to a limited amount of thrombin generation. The amount of thrombin produced is inadequate to support normal fibrin generation and in fact can be significantly inhibited by antithrombin. The signal becomes amplified when thrombin binds to platelets and initiates several positive feedback loops. Here we see the continuing role of platelets in this process, as well as the beginning of the feedback mechanism of thrombin. At the end of the amplification phase, the stage is set for the large burst of thrombin generation that is essential to stable clot formation.

In this phase, the assembled enzyme complexes on the platelet surface rapidly lead to the production of enough FIIa to support additional platelet activation. Additional platelet activation leads to ever-increasing amounts of FIIa and subsequent fibrin formation. So thrombin generation, is much more complex than originally thought.

What Does All of This Mean?
Cellular components place a large role in having blood clot and platelets function throughout the process. Most importantly, thrombin is generated in bursts and its feedback mechanisms are important in final clot formation. In the cell based coagulation cascade the extrinsic pathway operates on the theory that the tissue factor bearing cell initiates and amplifies coagulation, while the intrinsic pathway operates on the activated platelet surface to produce the burst of thrombin to stabilize the clot.



Diagnosing Factor Deficiences

Overview of the ISTH Conference

Guidelines: Do We Need Them? Should We Use Them? Are they Helpful?

June 2017 ISLH: Hawaii - Hot Topics

Factor XIII

Biological Variation in Coagulation

Previous Posts

This website contains information on products which is targeted to a wide range of audiences and could contain product details or information otherwise not accessible or valid in your country. Please be aware that we do not take any responsibility for accessing such information which may not comply with any legal process, regulation, registration or usage in the country of your origin.