By Dr. Maria Benito
The protective system of blood coagulation to prevent excessive blood loss after injury –in normal hemostasis– is based in a cascade of enzyme activations –proenzymes and procofactors– initiated by serine proteases with limited proteolysis, which results in the polymerization of fibrin and the activation of platelets, leading to a blood clot formation.
However, the blood clotting system –which can be triggered by two mechanisms: the tissue factor pathway that functions in normal hemostasis; and the contact pathway, which seems to act in host pathogen defenses– can lead to pathologic thrombosis –unwanted blood clots– that contributes to substantial disability and death in the developed world, being pulmonary embolism –with a 30% mortality rate within one month– the most damaging complication.
The tissue factor pathway –or Extrinsic Pathway– is triggered by a cell-surface protein known as tissue factor (TF). When cells expressing TF are exposed to blood, it immediately triggers the clotting cascade and the formation of blood clots.
The contact pathway –or Intrinsic Pathway– is triggered when plasma comes into contact with some artificial surfaces like glass test tubes or finely ground clay. This pathway does not contribute to normal hemostasis, but it is thought to participate in thrombotic diseases.
While hemostasis is the normal process by which the clotting cascade stops vascular damage by limiting blood loss after an injury, pathologic thrombosis triggers the clotting cascade in the lumen of a blood vessel, leading to the formation of a blood clot or thrombus that can obstruct the flow of blood. Furthermore, severe thrombosis can block the blood flow to a tissue, leading to ischemia and tissue death.
The extrinsic or TF pathway
The plasma clotting cascade involves a series of reactions leading to the activation of zymogens –precursors of enzymes– through limited proteolysis, which lead to the activation of the catalytic serine protease enzyme coagulation factor VIIa (fVIIa). This enzyme –which is a potent activator of coagulation–, contains two subunits: the trypsin-like serine protease catalytic subunit fVIIa, and the cofactor positive regulatory subunit TF.
The TF:VIIa complex is inhibited by the tissue factor pathway inhibitor (TFPI) –with two isoforms: TFPIα and TFPIβ, produced by megakaryocytes and endothelial cells, stored in platelets, and secreted upon platelet activation–, and is mostly associated with the microvascular endothelium, and some circulating. Moreover, the administration of heparin causes a rapid increase in the circulating levels of TFPI in plasma.
However, while the TF:VIIa complex is the crucial trigger for hemostatic responses in vivo, excessive initiation of coagulation via the extrinsic pathway can lead to thrombosis, consumptive coagulopathy –also known as disseminated intravascular coagulation, characterized by abnormally increased activation of procoagulant pathways–, or inflammation. An increase in the formation of this complex can be due to loss of vascular wall integrity, an increased TF expression, or amplified either levels or activity of fVII/fVIIa.
TF –also known as thromboplastin, coagulation factor III, or CD142– is a glycosylated, integral-membrane protein that does not need proteolysis to be activated; it is present in adventitial cells surrounding blood vessels and organ capsules, and is particularly abundant at anatomic sites where hemorrhage can result in disastrous consequences, such as kidney and brain, but usually absents in circulating blood cells and endothelial cells.
Atherosclerotic plaques also contain significant levels of TF, generally associated with monocytes, foam cells and smooth muscle cells. TF antigen may also be found in the acellular core of atheromas, most likely from necrotic cells. TF expression can also be increased with malignancy, leading to cancer-associated thrombosis. In these cases, neoplastic cells can express TF, but also tumour TF can be associated with infiltrating activated monocytes or stromal cells. Although still controversial, some studies pointed to the fact that neutrophils, eosinophils and platelets can express TF under certain circumstances, in a similar fashion of what cultured monocytes from peripheral blood and endothelial cells do under inflammatory conditions or hypoxia, which may play an important role in thrombotic diseases.
In order to propagate the clotting cascade, more activation of factors takes place. Thus, coagulation factor IX (fIX) and X (fX) are converted to activated factors fIXa and fXa, which assemble with their own protein cofactors –facot VIIIA (fVIIIa) in the case of fIXa; or fVa for fXa–, which leads to a large release of thrombin, that efficiently processes fibrinogen into fibrin via limited proteolysis. The next step is the formation of fibrin clots and further activation of platelets, contributing to both, the normal protective hemostasis, or the formation of a thrombus in pathologic activation of clotting.
In atherosclerosis, only a thin monolayer of endothelial cells separate blood from TF. Since myocardial infarction is considered to be triggered by the rupture of an atherosclerotic plaque in a coronary artery, as a result of that burst, TF is exposed to fVII/fVIIa within the blood. Myocardial infarction occurs if the process of coagulation activation is big enough to form an occlusive thrombosis within the coronary vessel. Epidemiologic studies suggests that elevated plasma fVII may be a risk factor for thrombotic disease; high levels of circulating fVIIa have also been found with angina, transient ischemic attacks, diabetes, uremia, and peripheral vascular disease.
The contact pathway
The role of contact activation pathway seems to be the generation of bradykinin –an inflammatory mediator involved in vasodilation, vascular permeability, pain, and neutrophil chemotaxis–, contribution to fibrinolysis, and inhibition of thrombin-induced platelet activation and angiogenesis.
This pathway of coagulation is initiated by activation of factor XII (fXII) –in a process involving kininogen (HK) and plasma pre-kallikrein (PK)– producing active factor XII (fXIIa), which activates PK to kallikrein, leading to thrombin generation and blood clots.
Specific cell surface proteins, extracellular nucleic acids, inorganic polyphosphate (polyP), misfolded proteins, glycosaminoglycans, and bacterial surface proteins are considered to be candidate for activators of the contact pathway. The plasma protease inhibitor, C1-inhibitor –an acute phase protein that increases under inflammatory conditions–, is a crucial regulator of the contact pathway, inhibiting fXIIa, fXIa, and other members of the complement cascade.
Dysregulation of the contact pathway –usually caused by deficiency of C1 inhibitor plays a role in patients with hereditary angioedema. Contact activation –due to continued generation of fXIIa and kallikrein– takes place during sepsis and other inflammatory response due to infections. This pathway also seems to contribute to thrombotic disorders, having been associated with atherosclerosis or myocardial infarction.
Date shows that severe fXI deficiency reduces the risk of stroke, while fXII deficiency decreases formation of arterial thrombi and protects the animals from ischemic brain injury in animal models of thrombosis, whereas RNA or polyP administration triggers pulmonary embolism.
There is a fine but complex relationship between hemostasis and pathological thrombosis. Classical anticoagulant drugs that interfere in both pathways –too much anticoagulation presents the risk of bleeding, and to little have the risk of thrombosis– are some of the most widely prescribed medications today. However, drugs that inhibit initiation of the contact pathway –by using antisense oligonucleotides to inhibit the biosynthesis of fXI or with a monoclonal antibody targeting the active site of fXIIa– may be more effective antithrombotics with little or no bleeding side effects.
Inhibition of the contact pathway as a method of anticoagulation not only carries less risk of bleeding than current therapeutics, it also has the potential to reduce the often damaging connections between coagulation and inflammation in human disease.
Role of Neutrophiles
Neutrophils have a critical role in the activation of various types of thrombosis in vivo. During infection and inflammation, neutrophils promote intravascular blood coagulation and thrombosis, contributing to cardiovascular diseases induced by thrombosis, such as myocardial infarction, stroke, and venous thromboembolism. It has been shown in different types of thrombosis –such as microvascular thrombosis during bacterial infection and carotid artery thrombosis–, that neutrophils amplify intravascular coagulation by stimulating the tissue factor-dependent extrinsic pathway via inactivation of endogenous anticoagulants, enhancing factor XII activation or decreasing plasmin generation. Additionally, studies in a mouse model have shown that the cooperation between platelets, monocytes and neutrophils cause the initiation and propagation of fibrin formation, therefore triggering deep vein thrombosis.
The interactions of neutrophils with activated platelets induce neutrophil extracellular traps, which are formed by externalization of decondensed nucleosomes and proteins. Dysregulation of this innate immune pathway may support the development of coagulopathies associated to sepsis. There are currently suggestions that these extracellular traps and nucleosomes –which are increased in thrombi and in vaso-occlusive pathologies– could be therapeutically used as targets for thrombosis prevention.
On the other hand, immunothrombosis involves the activation of intravascular blood coagulation and protective microvascular thrombosis to halt the circulating bacteria, and restrict tissue invasion, and survival in organs.
Interactions between activated platelets and activated neutrophils can activate the extrinsic pathway of coagulation, which increases fibrin formation initiated by tissue factor (TF), which can be silenced by different mechanisms under physiological conditions in order to prevent pathological obstruction in blood vessel due to blood coagulation. The anticoagulant tissue factor pathway inhibitor (TFPI) –which directly inhibits coagulation factors VIIa and Xa– controls the TF pathway, while neutrophil serine proteases degrade and inactivate TFPI.