Stephanie A. Smith, DVM, MS, DACVIM
Previous Coagulation Models
The "Y" Cascade Model
In the 1960's biochemists proposed a coagulation cascade model that has been modified and updated over the last several decades. This model, with which we are all very familiar, consisted of a cascade of steps where enzymes (often in conjunction with non-enzymatic cofactors) cleaved proenzyme substrates to generate the next enzyme in the cascade. The model was divided into the familiar "extrinsic" and "intrinsic" pathways. The "extrinsic" system was localized to outside (or "extrinsic from") the blood, and consisted of tissue factor (TF) and FVIIa. The "intrinsic" system was localized to within the blood (or "intrinsic to") and was initiated through the contact activation of FXII in conjunction with other proteins. Either pathway could activated FX to FXa, which in turn (with its cofactor FVa) could activate prothrombin to thrombin, which then cleaved fibrinogen to form fibrin. This latter portion was generally referred to as the "common" pathway. Deficiencies in the "extrinsic" and "common" pathways are identified using the prothrombin time (PT); while deficiencies in the "intrinsic" and "common" pathways are reflected with prolongation of the activated partial thromboplastin time (aPTT).
Deficiencies in the "Y" Cascade Model
While separating the various enzymatic processes of coagulation into the "Y" cascade is useful in furthering understanding of how coagulation processes occur, it is obvious that this model does not adequately explain the hemostatic process as it occurs in vivo. The "Y" cascade suggests that the "extrinsic" and "intrinsic" pathways operate as independent and redundant pathways, while clinical manifestations of individual factor deficiencies clearly contradict this concept. While deficiencies in the initial components of the intrinsic pathway (FXII, high molecular weight kininogen, or prekallikrein) cause marked prolongation of the aPTT, they are not associated with a tendency for abnormal bleeding. In contrast, deficiencies in downstream components of the intrinsic pathway (FVIII and FIX) result in the serious bleeding tendencies seen with hemophilia A and B, despite the fact that these patients have an intact "extrinsic" pathway. Similarly, FVII deficiency can be associated with bleeding, despite the presence of an intact "intrinsic" pathway.
In order to control hemorrhage in response to injury, hemostatic processes are required to create an obstruction at the site of injury. Lack of regulation of the hemostatic process has the potential to initiate coagulation (and consequently impede blood flow) at sites where no injury is present. Appropriate hemostasis consequently requires that coagulation be localized specifically at a site of injury. This localization is accomplished primarily via the contribution of membrane surfaces to coagulation processes.
Interactions of Coagulation Proteins with Membranes
The speed at which many of the enzymatic reactions in coagulation proceeds is profoundly affected by the presence of an appropriate membrane surface. These enzymatic reactions are enhanced by membrane binding of the participating proteins in part because localization to a membrane surface helps properly align the participating proteins. Tissue factor (TF) is the only coagulation protein that is permanently attached to the membrane surface. Other coagulation proteins contain gammacarboxyglutamic acid residues (Gla) that bind the protein to membrane surfaces through a calcium dependent mechanism. These Gla proteins (e.g., FVII, FIX, FX, prothrombin) require the vitamin-K cycle for proper development of their calcium- (and consequently membrane-) binding capabilities. The importance of membrane binding is consequently illustrated by the profound adverse impact of vitamin-K antagonists such as warfarin.
All cells are surrounded by lipid membrane bilayers which contain a large number of constitutively expressed membrane surface proteins. The composition and distribution of membrane phospholipid molecules is tightly controlled. In the inactive resting membrane state, neutral phospholipids are located on the external leaflet of the membrane, and the negatively-charge phospholipid phosphatydlserine (PS) and the neutral lipid phosphatidylethanolamine (PE) are localized to the inner surface of the membrane. This membrane asymmetry is essential and tightly controlled under normal conditions. When cells are activated or injured such as occurs with platelet activation and apoptosis of many cell types, cells actively shuffle the PS and PE to the outer membrane leaflet. This membrane phospholipid shuffling is controlled by a variety of enzymes (most notably "floppase" and "lipid scramblase") and occurs in response to increased concentrations of calcium in the cytosol.
Contribution of the Procoagulant Membrane
The expression of PS and PE on the cell surface has a profound impact on the procoagulant properties of the membrane surface. Although a full-understanding of how coagulation reactions occur on activated membrane surfaces has not yet been achieved, it is known that the presence of PS on the membrane markedly increases the speed of some coagulation reactions (>thousands of times faster). Less PS is required for maximum speed when PE is present. It is currently thought that Gla proteins preferentially bind to PS clusters on the membrane surface, and that PE aids in grouping PS into these clusters. The expression of PS (with and without PE) turns the cell membrane into a procoagulant surface. Because coagulation reactions occur very slowly on membranes that don't contain PS, resting cells are essentially incapable of supporting the coagulation cascade. Under normal physiologic conditions, cells outside of the injured area do not express a procoagulant membrane. Consequently, generation of coagulation enzymes is extremely slow, and insufficient to generate enough fibrin to form a clot.
Role of Microparticles
Microparticles are intact vesicles derived from cell membranes. They vary somewhat in size (2-20% of the size of a red blood cell). They arise when activated or apoptotic cells shed bits of membrane, and are primarily derived from endothelial cells, platelets, and monocytes. In certain disease states, microparticles from granulocytes and erythrocytes may also be important. Microparticles contain cell surface proteins similar to those found on their parent cell (e.g., ultra large vWF monomers on endothelial cell-derived microparticles, P-selectin on platelet-derived microparticles, TF on monocyte-derived microparticles) that can participate in coagulation reactions, especially when the microparticle expresses a procoagulant membrane. The contribution of microparticles to normal hemostasis in under intensive investigation currently, but their importance is demonstrated by the severe bleeding deficiency observed with Scott syndrome, an inherited deficiency of an enzyme that is required for production of microparticles.
Other Anticoagulant Properties of Cells
In addition to the resting membrane that does not support coagulation reactions, non-activated resting endothelial cells express a number of other anticoagulant properties via proteins expressed on their surface. These include heparan sulfated proteoglycans (HSPGs), thrombomodulin (TM), and tissue factor pathway inhibitor (TFPI).
Endothelial cells produce HSPGs, a small amount of which is expressed on the luminal surface in contact with the flowing blood. The HSPGs are a binding site for antithrombin (AT), which then is fully capable of inactivating thrombin which is produced in the vicinity of the HSPG. The inactivation of thrombin by HSPG-AT is similar to that by heparin-AT which we exploit clinically when administering soluble (non-membrane bound) forms of heparin.
Resting endothelial cells also express TM on their surface. Thrombin, once bound to TM, converts from procoagulant to anticoagulant because the thrombin-TM complex rapidly activates protein C. Activated protein C (with its cofactor protein S) then irreversibly cleaves FVa and FVIIIa, preventing their further participation in generation of additional new thrombin molecules. APC-PS also inactivates and important inhibitor of fibrinolysis (plasminogen activator inhibitor 1, or PAI-1) which upregulates lysis of any fibrin that is formed. Note that expression of TM is 100 fold higher in capillary endothelium as compared to endothelium in the major vessels. Therefore, any thrombin circulating in large vessels will be quickly extracted when the blood passes through a capillary.
TFPI on the endothelial cell surface prevents additional thrombin generation by acting as an upstream inhibitor of FXa and FVIIa. It irreversibly binds to FXa, then forms a quaternary complex between TFPI, FXa, FVIIa, and TF, preventing their further participation in the generation of additional thrombin molecules.
The Cell-Based Model
Our new understanding of hemostasis incorporates the role of cells. Evaluation of this model suggests that coagulation actually occurs in vivo in distinct overlapping phases. It requires the participation of two different cells types: a cell bearing TF, and platelets.
All evidence to date indicates that the sole relevant initiator of coagulation in vivo is TF. Cells expressing TF are generally localized outside the vasculature, which prevents initiation of coagulation under normal flow circumstances with an intact endothelium. Some circulating cells (e.g., monocytes or tumor cells) and microparticles may express TF on their membrane surface which under normal conditions is thought to be inactive. The exact role of this blood-borne TF is controversial. Some investigators believe that circulating TF is "encrypted" in that it contains an additional bond that most be cleaved for activity. Others believe that circulating TF is not fully active until the membrane surface on which it resides is not a PS-containing procoagulant membrane.
Once an injury occurs and the flowing blood is exposed to a TF-bearing cell, FVIIa rapidly binds to the exposed TF. The TF-FVIIa complex then activates small amounts of FIX and FX. The generated FXa binds to its cofactor FVa to form the prothrombinase complex, which subsequently cleaves prothrombin and generates a small amount of thrombin. Any FXa that dissociates from the membrane surface of the TF-bearing cell is rapidly inactivated by either TFPI and AT. The FXa generated is consequently effectively restricted to the surface of the TF-bearing cell on which it was generated. However, the FIXa generated can dissociate and move to the surface of nearby platelets or other cells. FIXa is not inhibited by TFPI, and much more slowly inhibited by AT than is FXa.
Note that because TF is always expressed in the perivascular space, any FVIIa that leaves the vasculature during normal plasma leakage will bind to TF and potentially initiate coagulation. The amount of protein leakage through a normal endothelium is generally restricted to smaller proteins. Most of the upstream coagulation proteins are relatively small (e.g., FVII: 50,000 daltons) whereas some of the downstream proteins are much larger (FV: 330,000 daltons, fibrinogen 340,000 daltons). This means that platelets and large proteins are sequestered from the extravascular space. Coagulation proceeds beyond the generation of the small amount of thrombin that occurs with initiation only when the injury allows platelets and larger proteins to leave the vascular space and adhere to the TF-bearing cells in the extravascular area.
Once a small amount of thrombin has been generated on the surface of a TF-bearing cell (the initiation phase), that thrombin is available for activation of platelets, and for activation of the necessary cofactors FV and FVIII and the generation of the enzyme FXIa. Binding of thrombin to platelet surface receptors causes extreme changes in the surface of the platelet, resulting in shuffling of membrane phospholipids to create a procoagulant membrane surface and release of granule contents that provide additional "fuel for the fire". Platelet alpha granules contain a large number of proteins and other substances which include raw materials for clotting reactions, agonists to induce further platelet activation, and calcium. Calcium may induce clustering of PS (which increases the procoagulant nature of the membrane), and promotes binding of coagulation proteins to the activated membrane surface.
Once a few platelets are activated in the amplification phase, the release of the granule contents results in recruitment of additional platelets to the site of injury. The propagation phase occurs on the surface of these platelets. Expression of ligands on their surface results in cell-cell interactions that leads to aggregation of platelets. FIXa that was generated in by TF-FVIIa in the initiation phase can bind to FVIIIa (generated in the amplification phase) on the platelet surface. Additional FIXa is generated by cleavage of FIX by FXIa (generated during amplification) on the platelet surface. Recall that FXa was generated during the initiation phase on the TF-bearing cell surface. Since this FXa is rapidly inhibited if it moves away from the TF-bearing cell surface, it can not easily reach the platelet surface. The majority of FXa must therefore be generated directly on the platelet surface through cleavage of the intrinsic tenase complex (FIXa-FVIIIa). This generated FXa then rapidly binds to FVa (generated by thrombin in the amplification phase) and cleaves prothrombin to thrombin. This results in a burst of thrombin generation with enough thrombin generated with enough speed to cleave fibrinogen, resulting in polymerization into a fibrin clot.
In order for coagulation to occur effectively, thrombin must be generated on the platelet surface (not just on the surface of the TF-bearing cell). This model adequately explains the bleeding defects observed with FXI, FIX, and FVIII deficiencies, because these proteins are required for generation of FXa (and subsequently thrombin) on platelet membranes.
Additional Controls to Terminate Coagulation
Once the fibrin/platelet clot has formed at the site of injury, coagulation must be limited to prevent widespread fibrin formation. Inevitably, some proteases diffuse away from the vicinity of the activated platelets and are carried downstream. As detailed above, the lack of procoagulant membrane on resting endothelial cells that are located away from the site of injury prevents efficient generation of thrombin by any FXa that flows through the vasculature. FXa and thrombin are also effectively inhibited by the endothelial cell surface associated anticoagulant proteins. In particular, thrombin generation is limited because activated protein C/protein S is a much better inactivator of FVa on the endothelial cell surface than on the platelet surface. This means that APC/PS is efficient at limiting thrombin generation on healthy resting endothelial cells, but not efficient at inhibiting generation of thrombin on activated platelets.
Difficulties Associated with Studying Coagulation
Studies of the processes of blood coagulation have primarily been limited to evaluating protein-protein interactions and platelet behavior in vitro. Collection of blood samples by definition involved entry into the vascular system with the likely local exposure of TF and the subsequent generation of thrombin. Collection of samples also necessitates some form of anticoagulation to preserve clotting ability until such time as the experiments are to be performed. The use of an anticoagulant (commonly citrate to chelate the calcium) potentially introduces a level of artifact into the subsequent studies. Limiting coagulation studies to the plasma portion of blood eliminates the extensive contribution of platelets to the coagulation process. While these studies have provided a wealth of knowledge regarding enzymatic capabilities, inhibitory interactions, and platelet function, they do not necessarily represent hemostatic processes as they occur in vivo.
Thromboelastography (TEG) is an in vitro method of evaluating the dynamics of clot formation in whole blood as it occurs over time. This invaluable tool includes the contribution of platelet cell surfaces and the potential impact of blood cells on hemostasis. While TEG generally still requires collection of blood into anticoagulant (and the consequent potential artifacts thus created), it is likely more representative of in vivo hemostasis than plasma based in clotting tests. Unfortunately, no in vitro testing system can adequately represent the contribution of an active vessel wall and its endothelium, flowing blood, and calcium.
A system developed by Barbara and Bruce Furie at Harvard has allowed for the study of real time hemostatic processes in the living animal. The intravital microscopy system allows for visualization of various components of hemostasis including calcium mobilization, platelet aggregation, tissue factor expression, and fibrin formation. Comparison of in vivo hemostasis in gene knockout mice (with specific components absent) to that in normal mice has provided invaluable information regarding the role of various participants in coagulation.
1. Hoffman M. Remodeling the blood coagulation cascade. J. Thromb Thrombol 2003;16:17-20.
2. Furie B, Furie BC. In vivo thrombus formation. J Thromb Haemost 2007; 5(Suppl 1):12-17.
3. Gailani D, Renné T. The intrinsic pathway of coagulation: a target for treating thromboembolic disease? J Thromb Haemost. 2007;5:1106-12.
4. Mackman N, Tilley Re, Key NS, The role of the extrinsic blood pathway of coagulation in hemostasis and thrombosis. Arterioscler Thromb Vasc Biol 2007;27:1687-1693.
5. Piccin A, Murphy WG, Smith OP. Circulating microparticles: pathophysiology and clinical implications. Blood Reviews 2007;21:157-171.
6. Polgar J, Matuskova J, Wagner DD. The P-selectin, tissue factor, coagulation triad. J Thromb Haemost 2005;3:1590-1596.
7. Sagripanti A, Carpi A. Antithrombotic and prothrombotic activities of the vascular endothelium. Biomed Pharmacother 2000; 54:107-111.
8. Verhamme P, Hoylaerts MF. The pivotal role of the endothelium in haemostasis and thrombosis. Acta Clin Bel. 2006;61:213-9.