The blood coagulation cascade is a remarkably complex biological process which has evolved over millions of years. This evolution has led to the development of an incredibly intricate yet coordinated system consisting of various components which interact in incredibly specific ways to yield a single final product; fibrin. The evolutionary complexity of the system is simultaneously its greatest strength, allowing for feedback regulation on a multitude of levels, and its greatest weakness, introducing more proverbial links in the chain which may ultimately fail. Yet it was through the discoveries of these supposed weak links which has led to most of our modern understanding of the functioning of the blood coagulation cascade. In 1936 researchers discovered that patients with hemophilia lacked a clotting protein necessary for proper clotting, which was later termed factor VIII (FVIII). The study of individuals with deficiencies in their clotting mechanism led to the discoveries of many other clotting factors including FV, FVII, FVIII, FIX, FX, FXI, and FXII. It is mainly through these discoveries that have led to the modern day understanding of the blood coagulation cascade, yet it apparent from the recent discoveries that this understanding is far from complete.
The blood coagulation cascade involves a series or “cascade” of zymogen activation reactions. At each stage a precursor protein (zymogen) is converted to an active protease by cleavage of one or more peptide bonds in the precursor molecule. The coagulation cascade consists of two pathways: the contact activation pathway, and the tissue factor pathway which lead to the ultimate activation of the final common pathway.
The activation of the contact activation pathway, formerly known as the intrinsic pathway, involves the mutual coordination of 4 key proteins which form what is known as the contact complex and consists of FXII, FXI, high molecular weight kininogen (HMWK) and prekallikrein (PK). It is the association of these proteins, in contact with an anionic surface, which leads to the activation FXII and corresponding contact pathway activation. PK, the zymogen of kallikrein, has the capability of inducing the direct activation of FXII and is also involved in the association of HMWK within the contact complex. HMWK is the main cofactor of the complex and contains binding sites for PK, FXI, and calcium along with a domain which is capable of interacting with hydrophilic and anionic surfaces. The role of HMWK in the contact activation pathway is related to its ability to coordinate the FXI-PK-FXII complex together resulting in the activation of FXII. Activated FXII (FXIIa) is able to directly activate FXI (FXIa) in the presence of HMWK which in turn is able to activate FIX (FIXa), an event in which calcium and platelets, or rather a phospholipid surface are primary cofactors. FIXa combines with its primary cofactor activated FVIII (FVIIIa) to form a membrane (phospholipid)-bound complex known as the Xase ternary complex, which results in the direct activation of FX and corresponding common pathway.
Research has recently shown that the tissue factor pathway is the primary pathway of fibrin formation, while the contact activation pathway acts primarily as an amplifier of cascade activation and fibrin generation. The activation and sustainment of the tissue factor pathway involves the coordination of a multitude of proteins and cofactors including FVII, calcium, tissue factor pathway inhibitor (TFPI), tissue factor (TF), and a phospholipid surface. Human coagulation FVII is a single chain, glycoprotein which circulates in normal human blood and is the main activator of the tissue factor pathway. Cleavage of a single peptide bond results in a structural change which activates the zymogen transforming it into a potent vitamin K-dependent serine protease. This structural change also allows for the protein to effectively bind to its cofactor, TF. The distribution of TF is carefully coordinated in a hemostatic layer, with the ability to induce coagulation upon the disruption of the vascular endothelium. TF forms a stable complex with FVIIa, and this complex is capable of directly initiating the common coagulation pathway via the activation of FX or indirectly through the activation of FIX which in turn is able to activate FX. TF is most effective as a procoagulant when incorporated into phospholipid membranes, a surface which is typically provided by platelets.
Calcium has been shown to play a key role as a cofactor in the generation and activity of several vitamin K-dependant serine protease coagulation factors, including FVIIa. A high-affinity calcium binding site in the protease domain of FVII has been shown to mediate the interaction between TF and FVIIa. Conversely, the γ- carboxyglutamic acid (Gla) domain of FVIIa contains several low affinity calcium binding moieties which aid in the interaction of FVIIa and phospholipid surfaces. In most instances, calcium binding leads to a necessary conformation change which allows these factors to effectively “dock” on a surface and exhibit their cleavage site. Without the binding of calcium, in calcium depleted medium, it is believed that this Gla domain slightly unfolds exposing a highly negatively charged domain to the media which is capable of interacting with a positively charged surface. Although groups have shown the capability of FVII to bind to such positively charged surfaces in calcium depleted environments, such as citrated plasma, the mechanism by which this occurs, specifically the material properties which affect this process, are not well understood. TFPI is the main inhibitor of the FVIIa-TF complex, and is an important feedback regulator of tissue factor pathway activation.
The activation of FX by either FIXa or the FVIIa-TF is the initial step in the activation of the final common pathway. Immediately after activation FXa complexes with FVa to form a complex, known as the prothrombinase complex, that is capable of converting the zymogen prothrombin (FII) to its enzymatic analogue thrombin (FIIa). The ability of this prothrombinase complex to convert prothrombin to thrombin is highly dependent on both calcium and a phospholipid surface, which is typically provided by platelets. Thrombin catalyzes a multitude of key reactions including platelet activation, activation of FV, FVIII, FXI, and FXIII along with directing the disassociation of FVIII from vWF, and the conversion of fibrinogen to fibrin. Although the initial amount of thrombin formation is unable to produce a sufficient amount of fibrin to stabilize the formed platelet plug, the reactions it sets in place in combination with the sustained activation of FX lead to a subsequent thrombin burst which results in substantial fibrin formation. Fibrin strands form spontaneously after conversion, which are subsequently cross-linked by FXIIIa resulting in a matrix which entrap platelets and erythrocytes (red blood cells) to produce a stabilized hemostatic plug at the wound site.