Classic cascade mechanism of coagulation vs. cell-based models of thrombin generation

In the mid 1960s two different investigators conceived an original concept of the coagulation mechanisms triggered as a ‘cascade’ of proteolytic reactions acting as biological amplifiers.^1,2 According to those classic concepts, coagulation mechanisms were activated through intrinsic and extrinsic pathways converging on a common pathway that will finally activate prothrombin into thrombin that would cause soluble fibrinogen to become polymerized into a solid fibrin clot (Fig. 36.1).

Fig. 36.1 The classic coagulation cascade. The intrinsic coagulation pathway is initiated when blood gets in contact with a foreign surface. The extrinsic pathway is initiated after tissue factor (TF) is exposed at sites of vascular injury. The two pathways converge at the activation of FX to FXa and a common pathway that leads to thrombin formation. Ovals indicate reactions that are accelerated by anionic phospholipids (PL) exposed on activated platelet membranes.

Modern concepts have integrated the classic stepwise cascade into a more comprehensive scheme in which blood coagulation is initiated by cellular components in vivo.³ Actual models contemplate the implication of cellular and enzymatic mechanisms in three differentiated steps: Initial activation, propagation and thrombin generation (Fig. 36.2). Fibroblasts, smooth muscle cells of the vascular wall, activated monocytes or endothelial cells, or even platelets are potential cellular sources of TF.^4,5 According to current knowledge, the activation of the coagulation is initiated at the very moment in which TF is exposed on cellular surfaces. TF is a transmembrane protein that binds plasma factor VII/VIIa. In a first step, the TF exposed on damaged vascular areas forms a complex with FVII/FVIIa. FVIIa is present in reduced amounts in circulating blood. The TF : FVIIa complexes will activate FIX and FX. FXa generated will activate small quantities of prothrombin (FII) into thrombin (FIIa). Small amounts of thrombin generated in this short loop will be able to promote further activation of the coagulation by further activating FV and FVIII in the presence of platelet phospholipids and FIX.³ During the propagation step FIXa bound to FVIIa on the surface of activated platelets form the tenase complex that will further activate FX into FXa. FXa binds to FVa on the phospholipid surface provided by activated platelets and form the prothrombinase complex. During the final step of the cell-based model, the prothrombinase complex will further magnify the generation of thrombin facilitating the conversion of prothrombin into thrombin. Large amounts of thrombin generated during the final step will convert fibrinogen into fibrin. Thrombin itself activates FXIII thus stabilizing the fibrin formed.

Fig. 36.2 The cell-based model of coagulation emphasizes that tissue factor (TF) exposed on cell surfaces is the key event for the activation of coagulation under physiological circumstances. TF can be provided by fibroblasts, smooth muscle cells of the vascular wall, activated monocytes, endothelial cells, platelets or transported by microvesicles (MV). TF expressed on damaged or activated cellular surfaces forms a complex with FVII-FVIIa. This complex will activate FX to FXa and FIX to FIXa. FXa stays in the vicinity of the TF-bearing cell and, in the presence of FVa, converts a small amount of prothrombin (FII) to thrombin (FIIa) on the cell surface. The small amount of thrombin generated will be sufficient to activate platelets and FVIII, FV, and FXI. FVIIIa, FVa, and FXa occupy sites on the activated cell surface (activated platelets). FIXa, activated by TF-FVIIa, does not remain on the TF cell, but converts FX to FXa on the activated cell surface. FXIa acts to boost FIXa generation on the activated platelet, increasing FXa and subsequent thrombin generation. The intrinsic pathway may also take place on cellular surfaces, but its contribution to physiological coagulation is limited.

(Adapted from references 3–5).

Molecular mechanism

The majority of enzymes in the coagulation cascade are members of the same group of proteins called serine proteases (Table 36.1). They exhibit their function by limited proteolysis of peptide bounds of their substrates. The cascade design of the coagulation system allows a great amplification of signal in each step, since the enzymes activate more than one molecule of substrate. However, the enzymes by themselves do not have high catalytic efficiency and therefore the assembly of enzyme complexes is of crucial importance. This is for instance demonstrated by the bleeding diathesis experienced by hemophilia A patients, where the co-factor FVIII is deficient. In the enzyme complexes the activated membrane, exposing negatively charged phosphatidyl-serine plays a key role. Both the substrate and enzyme bind to the membrane concentrating the proteins at the surface. The correct orientation of the proteins on the cell surface facilitates the interaction. The importance of the membrane is shown in studies of the prothrombinase complex where the addition of phospholipid membrane gives an about 1000-fold increase in catalytic efficiency. Addition of co-factor FVa gives an additional increase and thus the prothrombinase complex is 10⁶ times more efficient than the enzyme alone.⁶

Table 36.1 Procoagulant and anticoagulant factors

Vitamin K-dependent proteins and their binding to the membrane

The vitamin K dependent plasma proteins are a group of Gla domain containing proteins that require vitamin K for their synthesis⁷ (Table 36.1). The Gla domains contain 9–12 Glu residues that are γ-carboxylated in a post-translational process that require vitamin K. The γ-carboxylated Glu residues (Gla residues) mediate the binding of Ca²⁺ ions and are crucial for the membrane interaction. Thus, in patients on treatment with vitamin K antagonist (coumarin) the γ-carboxylation is impaired leading to loss of calcium and membrane binding. Due to high sequence similarity, the fold and phospholipid binding mechanism are thought to be similar for all the Gla domains. Although the sequence of the Gla domains are very similar for all the vitamin K zymogens, their affinities for the membrane surface vary 100 to 1000-fold. It has been suggested that this is caused by amino acid sequence differences in just a few positions.⁸

SERPINs: a family of coagulation inhibitors

An important proportion of the natural anticoagulants exert their inhibitory action through inhibition of the enzymatic action of procoagulant serine proteases. This family of inhibitory proteins is called SERPINs (SERine Protease INhibitors). Antithrombin (SERPINC1), heparin co-factor II (SERPIND1), protein C inhibitor (SERPINA5) and PAI-1 (SERPINE1), are representative members of this family of inhibitors (Table 36.1). Although their primary sequence similarity is low, these proteins have a conserved three-dimensional structure that is crucial for their function in the regulation of hemostasis, thrombosis and fibrinolysis.¹¹ Their mechanism of action can be compared to a mousetrap, where the serine protease to be inhibited is lured into an irreversible binding leading to a distortion of the enzyme and loss of activity.¹² The inhibition is irreversible and one molecule of serpin can only inhibit one molecule of serine protease.

Antithrombin

Antithrombin (AT) is a α₂-globulin, member of the serpin family of protease inhibitors (SERPINC1), synthesized in the liver and is the major inhibitor of blood coagulation. Human AT is a single chain glycoprotein of 432 aminoacids with a reactive site cleaved by target enzymes located at Arg393-Ser 394. Antithrombin is a very powerful inactivator of thrombin (FIIa) and FXa, but does also inhibit FVIIa, FIXa, FXIa and FXIIa to a lesser extent.¹³ Antithrombin has a molecular weight of 58 200 kDa. There are two isoforms, α and β, of circulating AT. The α represents 90–95% and the β, 5–10% of the total. Under physiological conditions AT circulates in a form that expresses low inhibitory affinity. AT possess a binding site for heparin, being the β isoform the fraction with a higher affinity for heparin.¹⁴ Under physiological conditions heparin sulfates present on the surface of endothelium will provide a background level of activation on AT. Interaction of specific glycosaminoglycan sequences with the specific heparin-binding domain induces a conformational change in AT and accelerates by an order of a thousand times its inhibitory action on activated serin proteases.¹⁵ Inactivation of thrombin (FIIa) by AT produces thrombin–antithrombin complexes (TAT) that can be determined and used as a surrogate marker of the activation of the coagulation. The anticoagulant action of conventional heparins, low molecular weight heparins, and pentasaccharide is mediated by AT. Congenital or acquired deficiencies of AT, may reduce the therapeutic action of the previous anticoagulants. In addition to its anticoagulant action AT has an important anti-inflammatory effect that seems closely dependent on its ability to bind to endothelial glycosaminoglycans.

Protein C system

The protein C system restricts the propagation phase of the coagulation by down-regulation of the FV and FVIII. This regulation requires the assembly of the enzyme, activated protein C with the substrate FV or FVIII on membrane surfaces. As for the procoagulant enzymes, full anticoagulant effect of activated protein C requires presence of co-factors. In the case of FVa-degradation, protein S is the co-factor, whereas in the inactivation of FVIII two proteins function as co-factors, protein S and the intact form of FV.

Protein S

Protein S is a 70 kDa large vitamin K-dependent protein that circulates in the blood in a concentration of 20–25 mg/ml. More than 60% of the protein S circulates in blood bound to a complement regulator protein called C4b-binding protein (C4BP).²⁰ Protein S is mainly synthesized in the liver but synthesis has also been observed in endothelial cells, by testicular Leydig cells and by osteoclasts.²¹ Protein S has the following domain structure: an N-terminal vitamin K-dependent Gla domain, a thrombin-sensitive region (TSR), four EGF-like domains and two laminin G-type (LamG) domains (also called the SHBG-like domain). Like the other vitamin K-dependent blood proteins, the Gla domain of protein S mediates the membrane binding. Of the vitamin K-dependent proteins, protein S has the highest affinity for the membrane surface.⁸ The TSR is not found in the other vitamin K-dependent proteins. The region is sensitive to thrombin cleavage and cleavage leads to loss of APC co-factor function.²² Factor Xa has also been reported to cleave in the TSR, with the same result.

Tissue factor pathway inhibitor TFPI

TFPI, also known as lipoprotein-associated coagulation inhibitor or extrinsic pathway inhibitor, is a Kunitz-type inhibitor with a molecular weight of 34 kD.²⁶ The molecule possesses an acidic amino terminus, three Kunitz-type domains and a basic carboxy terminus. Circulating TFPI is synthesized by endothelial cells and smooth muscle cells.²⁷ Approximately 10% of the blood TFPI is present in platelets. TFPI neutralizes FVIIa-TF complexes, but is also capable of neutralizing FVIIa and FXa, though at a much slower rate. In fact, the inhibitory action of TFPI on FVIIa-TF complexes requires certain levels of FXa. In experimental animals, administration of TFPI reduces mortality in a model of septic shock induced by Escherichia coli.²⁸

Abnormal levels of TFPI or mutation in the respective gene are not strongly associated with thrombophilia. Treatment with exogenous hormones has a profound lowering effect on TFPI levels in women, with lower levels in oral contraceptive users than in premenopausal non-users. Levels of TFPI are strongly reduced levels in patients with abetalipoproteinemia, though they do not seem to be exposed to an enhanced risk of thrombosis. Recent studies indicate that low levels of TFPI, especially low TFPI-free and total antigen in plasma, may constitute a risk factor for DVT.²⁹

Protein Z-dependent protease inhibitor

Protein Z is a vitamin K-dependent protein with a molecular weight of 62 kD, plasma concentration ranging from 2 to 3 µg/ml and with an approximate half-life of 2.5 days. Although the structure of protein Z contains a Gla domain it does not possess protease activating action on other coagulation factors. Protein Z promotes FXa inhibition induced by ZPI.³⁰ Protein Z-dependent protease inhibitor (ZPI) is another member of the serpin family (SERPINA10) with known inhibitory action on FXa and FXIa.³¹ Heparin enhances the interaction of ZPI with FXIa.

The prothrombotic phenotype associated to protein Z-ZPI deficiency is controversial. Knock out mice for the protein Z gene do not seem to develop a thrombotic tendency unless there is another associated condition such as factor V Leiden.³² It has been postulated that thrombotic potential of the protein Z and ZPI deficiencies studies in humans question the thrombotic relevance of both ZPI and PZ deficiencies except when they act in combination with other prothrombotic factors.³⁰