The Cascade-Waterfall Model of Thrombin Generation

The cascade-waterfall hypotheses of intrinsic coagulation was first proposed in 2 landmark articles in 1964 by Macfarlane, and by Davie and Ratnoff. In subsequent models based on this scheme, the process of thrombin generation is the result of amplification of a procoagulant signal initiated by conversion of fXII to fXIIa, followed sequentially by activation of the enzyme precursors fXI, fIX, fX, and prothrombin ( Fig. 1 A ). At the time this model was proposed, it was recognized that much of the cascade could be bypassed through a process involving fVIIa (see Fig. 1 A), but the importance of this was not clear. The scheme in Fig. 1 A depicts the major enzyme reactions that contribute to plasma clotting in the activated partial thromboplastin time (aPTT) and prothrombin time (PT) assays used in clinical practice. There are 2 triggering mechanisms that converge at the level of fX activation. Activation through the intrinsic pathway (see Fig. 1 A, yellow arrows) is assessed by the aPTT assay, and is initiated by a process involving fXII called contact activation (discussed later). In the PT assay, coagulation is triggered through the extrinsic pathway by adding tissue extracts to the plasma that contain tissue factor (TF), a cofactor for fVIIa.

( A ) The waterfall/cascade model of coagulation: the proteolytic reactions leading to thrombin generation during plasma coagulation initiated through the intrinsic ( yellow arrows ) and extrinsic (fVIIa/tissue factor [TF]) pathways. The zymogen (precursor) forms of plasma trypsin-like proteases involved in coagulation are represented by the black Roman numerals, and their active protease forms by Roman numerals followed by a lowercase “a.” PK is the zymogen of the protease alpha-kallikrein. Protein cofactors are shown in red ovals, and reactions requiring calcium (Ca ²⁺ ) or phospholipid (PL) are indicated in blue. A feature of this model is that fXa and thrombin formation can be initiated through 2 distinct pathways. In the PT assay, coagulation is initiated by addition of the cofactor TF to plasma. TF in complex with fVIIa forms the extrinsic pathway of coagulation, which activates fX to fXa. In the activated partial thromboplastin time, addition of a negatively charged substance (usually a purified earth) to plasma triggers activation of fXII, and sets off the series of enzymatic reactions referred to as the intrinsic pathway of coagulation. The intrinsic pathway protease fIXa is an activator of fX in this model, bypassing the need for fVIIa/TF. fXa converts prothrombin to the protease thrombin, which then induces coagulation of plasma by converting fibrinogen to fibrin. ( B ) fVIIa/TF-initiated model of coagulation. The model presents the current understanding of the major plasma protease-substrate interactions during thrombin generation in vivo. The nomenclature and symbols are the same as those used in ( A ). Coagulation is initiated by exposure of the fVIIa/TF complex to blood, leading to the activation of fIX and fX. Early in the process, fXa converts prothrombin to thrombin to initiate coagulation, whereas fIXa generates more fXa to sustain the process. In this scheme, fXIa may contribute to thrombin generation by converting additional fIX to fIXa. Because fXII deficiency does not cause a hemostatic abnormality, it is thought that fXI is converted to fXIa by a protease other than fXII. For example, fXI can be activated by thrombin ( green arrow ). In contrast with the cascade/waterfall model in ( A ), here the intrinsic pathway ( yellow arrows ) triggered by initial fVIIa/TF-mediated thrombin generation is not part of mechanism for initiating coagulation, but functions to sustain fIX activation, and ultimately thrombin generation, to maintain the integrity of a clot over time. ( C ) The kallikrein-kinin (contact activation) system. When blood is exposed to a variety of surfaces and natural compounds (often with a net negative charge), the plasma zymogens fXII and PK bind to the surface and reciprocally activate each other. Binding of PK is facilitated by the cofactor high-molecular-weight kininogen (HK). The process is probably triggered by traces of fXIIa or alpha-kallikrein present in plasma. Alpha-kallikrein can cleave HK to release the proinflammatory peptide bradykinin. The kallikrein-kinin may have several functions, including contributing to the innate immune response to microorganism invasion. Although fXIIa can initiate thrombin generation through the intrinsic pathway of coagulation ( yellow arrows ) by activating fXI, the kallikrein-kinin system does not seem to be required for hemostasis. Humans and other animals lacking fXII, PK, or HK do not have a bleeding disorder. However, there is mounting evidence that contact activation–induced coagulation through the intrinsic pathway may contribute to growth of pathologic clots during thrombosis. The nomenclature and symbols are the same as those used in ( A ). α-Kal; alpha-kallikrein.

The sequential steplike nature of the coagulation cascade implies that complete deficiency of any component would break the reaction chain and cause bleeding, but this is not what is observed in clinical practice. Absence of fIX or its cofactor fVIII causes the severe bleeding disorder hemophilia, implying an important role for the intrinsic pathway in hemostasis. However, complete fXII deficiency, despite causing a marked prolongation of the aPTT, does not cause abnormal bleeding, whereas fXI deficiency causes a mild hemorrhagic disorder that involves tissues distinct from those commonly affected in fIX or fVIII deficiency. These observations indicate that the classic intrinsic pathway does not accurately describe the manner in which its components contribute to hemostasis, and have led to revisions in the models that are more in line with clinical phenotypes.

Tissue Factor–initiated Thrombin Generation: The Role of Factor XI

It is the current consensus that thrombin generation at a site of vascular injury is initiated primarily by fVIIa in complex with TF ( Fig. 1 B), an integral membrane protein expressed on cells underlying blood vessel endothelium. FVIIa/TF initiates coagulation by activating fX, as in the PT assay (see Fig. 1 A), and also activates fIX, which sustains fXa and thrombin production. The protease precursors (prothrombin, fVII, fIX, and fX) and cofactors (TF, fVa, and fVIIIa) shown within the gray area in Fig. 1 B form the core mechanism for thrombin generation in almost all vertebrate organisms. Total deficiency of any one of these proteins results in a severe bleeding disorder or is not compatible with life. This core mechanism is the target of all currently approved anticoagulants. Thus, it is to be expected that use of these compounds will increase bleeding risk.

In addition to the core proteins, mammals have fXI, the precursor of the protease fXIa (see Fig. 1 B). Severe fXI deficiency may cause excessive bleeding with trauma, particularly if tissues with robust fibrinolytic activity, such as the oropharynx or urinary tract, are involved. However, unlike fIX deficiency, severe spontaneous bleeding is not common in fXI deficiency. Furthermore, bleeding is highly variable, and some fXI-deficient individuals are asymptomatic despite marked abnormalities in the aPTT. Although fXI is activated by fXIIa in the cascade-waterfall model (see Fig. 1 A), the absence of abnormal bleeding in fXII-deficient individuals indicates that other mechanisms for fXI activation must exist. For example, thrombin generated early in coagulation can activate fXI (see Fig. 1 B, green arrow).

In the scheme shown in Fig. 1 B, fXIa sustains thrombin generation by activating fIX, rather than contributing to initiation of thrombin formation, as in the cascade-waterfall model and the aPTT assay. A key feature of the newer model is that there are 2 mechanisms for fIX activation, explaining the differences in bleeding phenotypes associated with fIX and fXI deficiencies. In the absence of fXI, fIX still contributes to thrombin production because it can be activated by factor VIIa/TF. Clinical observations suggest that factor VIIa/TF is probably the more important mechanism for fIX activation. Along similar lines, the different phenotypes associated with fXI and fXII deficiency are explained by the presence of more than 1 mechanism for fXI activation. Current models of thrombin generation often do not include a role for fXII, based largely on the observation that fXII deficiency is not associated with a bleeding diathesis. However, as discussed later, it does not necessarily follow that fXIIa does not contribute to thrombin generation under any circumstances.

The Kallikrein-Kinin System and Contact Activation

The plasma protease precursors fXII and PK and the cofactor high-molecular-weight kininogen (HK) form the plasma kallikrein-kinin system. fXII was first identified as a plasma constituent missing in a patient with a very long aPTT, but without abnormal bleeding. Subsequent work identified PK and HK as necessary for normal fXII activation in the aPTT. When plasma is exposed to artificial surfaces or anionic substances, fXII and PK undergo reciprocal conversion to fXIIa and alpha-kallikrein by a process called contact activation ( Fig. 1 C). HK serves as a cofactor during this process. In the aPTT assay, a purified earth such as silica or Celite is used to induce contact activation, with the resulting fXIIa promoting thrombin generation by activating fXI. The absence of bleeding symptoms in individuals lacking fXII, PK, or HK shows that these proteins are not required for hemostasis, either because they do not normally contribute to thrombin generation or because other mechanisms, such as thrombin-mediated feedback activation of fXI (see Fig. 1 B), compensate for their absence. The kallikrein-kinin system is thought to contribute to several homeostatic and host-defense mechanisms, including inflammation and the innate immune response to microorganisms. It is also clear that fXIIa and fXIa, despite their limited roles in hemostasis, are required for thrombosis in experimental animal models. If these proteins prove to be important contributors to thrombosis in humans, inhibiting them may produce an antithrombotic effect without significantly affecting hemostasis. The current understanding of the intrinsic pathway proteins in thrombosis in animal models and in human populations is discussed later.

Animal Models of Thrombosis

Mice lacking individual components of the intrinsic pathway, including the kallikrein-kinin system, have been tested for resistance to thrombus formation. FIX-deficient mice have a significant bleeding disorder (the murine equivalent of hemophilia B), and are resistant to thrombus formation induced by injuring blood vessels with concentrated ferric chloride (FeCl ₃ ). In contrast, mice lacking fXI or fXII do not have obvious defects in hemostasis. Despite this, counterintuitively, both fXI-deficient and fXII-deficient mice are as resistant to FeCl ₃ -induced thrombosis as are fIX-deficient mice. The findings have been corroborated using a variety of other models and methods for inducing thrombosis, and clearly indicate that an anticoagulant (antihemostatic) phenotype is not a prerequisite for an antithrombotic effect, at least under experimental conditions. More recent work has shown that mice lacking PK or HK are also resistant to injury-induced thrombus formation. Overall, these data imply that a process similar to classic contact activation may be driving thrombus formation through the intrinsic pathway in the mouse models.

Studies in primates have also shown roles for fXI and fXII in experimental thrombosis. This work used inhibitory antibodies or antisense oligonucleotides (discussed later) to produce transient deficiency states. Thrombus formation was studied in prothrombotic collagen–coated or TF-coated vascular grafts inserted into temporary arteriovenous shunts in olive baboons ( Papio anubis ). In these studies the antithrombotic effect of fXI inhibition seemed to be greater than that of fXII inhibition. A reduction in thrombus formation was detectable with as little as a 50% reduction in plasma fXI level, with the maximum effect achieved at less than or equal to 20% of normal plasma concentration.

Although the data from the animal studies support the notion that the intrinsic pathway contributes to thrombosis, there are limitations to the preclinical analyses that must be considered. Because the animal species tested do not have natural propensities to form arterial or venous thrombi in the same manner as humans, vessels must be injured in some manner to produce a thrombus. Injury-induced thrombosis in an otherwise healthy, and usually young, animal may not mimic formation of thrombi in the diseased vessels of older humans.

Factor IX and Factor VIII in Thrombosis in Humans

FIX is the precursor of the protease fIXa, and activated fVIII (fVIIIa) is its cofactor. The severe bleeding disorders associated with deficiency of fIX (Hemophilia B) or fVIII (Hemophilia A) show their importance to hemostasis. There is a consistent correlation between plasma levels of fVIII in complex with von Willebrand factor (vWF) and risk for venous and arterial thrombosis in humans. Supporting these observations, individuals with blood group O, who have 25% to 50% lower vWF and fVIII levels than individuals with a non-O blood group, are at lower risk for venous and arterial thrombosis. High plasma levels of fIX are associated with modestly increased risks for venous thromboembolism (VTE), myocardial infarction (MI), and stroke. These findings are supported by the impression that hemophiliacs have a lower risk of arterial thrombosis than nonhemophiliacs.

Factor XI in Thrombosis in Humans

For fXI levels, the strongest correlations are with risk for VTE and ischemic stroke. The 10% of subjects in the Leiden Thrombophilia Study with the highest plasma fXI levels had a 2-fold higher risk of VTE compared with the lower 90%, a result supported by recent data from the Longitudinal Investigation of Thromboembolism Etiology (LITE) cohort. Consistent with these findings, fXI deficiency was associated with a reduced incidence of VTE in a study from Israel, where severe deficiency is present in 1 in 450 individuals. High plasma levels of fXI or fXIa were associated with increased risk for ischemic stroke in a study by Yang and colleagues, in the Atherosclerosis Risk in Communities (ARIC) study, and in the Risk of Arterial Thrombosis In relation to Oral contraceptives (RATIO) study. Similarly, severe fXI deficiency was associated with a reduced incidence of stroke.

A role for fXI in MI is not as clear as for VTE or stroke. Plasma fXI levels correlated with MI risk in men in the Study of Myocardial Infarction Leiden (SMILE), and were higher in women with coronary disease than in women without it in a group undergoing cardiac catheterization. However, fXI was not a risk factor for MI in the ARIC or RATIO studies, and fXIa levels were not linked to coronary disease in the second Northwick Park Heart Study (NPHS-II). The incidence of MI in 96 individuals with severe fXI deficiency was similar to the expected incidence for age-matched controls. The data are in line with recent work by Siegerink and colleagues showing that an increased level of fXIa correlated more closely with risk for ischemic stroke than for MI in young women, and suggest that the contribution of fXI to thrombus formation is greater in some vascular beds than in others.

The Kallikrein-Kinin System in Thrombosis in Humans

Data supporting a role for fXII in VTE, stroke, or MI in humans are weak, and there is insufficient information to assess PK and HK. Note that congenital deficiency of C1-inhibitor (C1-INH), the major plasma regulator of fXIIa and alpha-kallikrein, causes hereditary angioedema, and does not seem to predispose to thrombosis. FXII-deficient individuals are not protected from VTE, and no differences in VTE incidences were noted across a range of fXII levels in LETS and LITE study participants. Data on fXII in arterial thrombosis are conflicting. FXII deficiency does not seem to protect individuals from stroke. Plasma levels of fXIIa were inversely correlated with stroke risk in NPHS-II, but were directly correlated with it in the RATIO study. However, plasma fXII levels did not correlate with stroke risk in either study, or in the ARIC study. In NPHS-II, increased fXIIa levels measured by specific enzyme-linked immunosorbent assay was associated with increased risk of MI, but fXIIa level measured as a complex with C1-INH indicated the opposite effect. In the RATIO study, fXIIa, fXII, and PK levels were not associated with MI, and risk of coronary events did not correlate with fXII in the ARIC study. In addition, data from the SMILE cohort showed an inverse relationship between fXII levels and cardiovascular disease. Endler and colleagues also noted an inverse association between fXII and death from ischemic heart disease, although the relationship did not hold for severe fXII deficiency (<10% of normal), for which the risk was comparable with that of the population mean.

The Intrinsic Pathway and Thrombosis in Humans: Summary

Although it is difficult to draw firm conclusions from the complicated human epidemiologic data, it seems reasonable to conclude that the contribution of fXII to VTE, stroke, and MI in humans is probably smaller than for fXI and fIX. Although this conclusion conflicts with data from mouse models, in which fXII is a major contributor to thrombus formation, it is in reasonable agreement with results obtained in primates. It is possible that feedback activation of fXI by thrombin (see Fig. 1 B) is stronger in primates than in mice, with fXIIa serving a smaller role in fXI activation. Another possibility is that a greater degree of fXIIa inhibition is required to produce an antithrombotic effect comparable with what is seen with fXIa inhibition in primates. These observations notwithstanding, there are situations in which fXIIa is likely to be a major driver of thrombosis in humans. For example, contact activation is triggered when blood is exposed to artificial surfaces during cardiopulmonary bypass and extracorporeal membrane oxygenation (ECMO), requiring full anticoagulation with heparin to prevent thrombotic complications.