Natural anticoagulants and fibrinolysis

This chapter is relevant to Section Q1(v) of the 2023 CICM Primary Syllabus, which expects the exam candidates to be able to "describe the mechanisms of preventing thrombosis including endothelial factors and natural anticoagulants". It probably also has some relevance to the "fibrinolysis" part of Section Q1(iv), "describe the process and regulation of haemostasis, coagulation and fibrinolysis". Of course, one might argue that this topic has minimal meaning for the intensivist, as in the critically ill these natural mechanisms of preventing thrombosis are either totally absent or so dysfunctional that they are not to be trusted, and everybody is on some combination of chemical and mechanical VTE prophylaxis. Still, people have to answer SAQs like Question 10 from the first paper of 2019, where for 50% of the marks we had to "discuss the role of naturally occurring anticoagulants in preventing clot formation in-vivo". Discuss, they asked, as if this were some chat over coffee, rather than a stressful barrier exam. Fortunately, the events of haemostasia - though they are simultaneous and interlinked - when beheld as sequential processes present themselves as a handy structure for an answer such as this. What follows is an attempt to use that structure to produce a short summary for revision purposes.

  • Factors that prevent the initiation of thrombosis
    • Normal endothelium does not contain any clotting cascade activators (eg. TF)
    • Endothelial glycocalyx contains antithrombotic molecules:
      • Heparin-like oligosaccharides
      • Antithrombin-III
    • Blood flow produces shear stress which opposes regional clotting factor concentration and platelet aggregation
  • Factors that prevent primary haemostasis
    • Extrinsic pathway inhibitors:
      • Tissue factor pathway inhibitor (TFPI) antagonises the TF-FVIIa complex and prevents extrinsic pathway activation
    • Platelet activation/aggregation inhibitors:
      • Prostacyclin (PGI2), via GS-protein-coupled receptor
      • Nitric oxide (less iCa2+, also inhibits TXA2 receptor)
      • Fibrin degradation products (bind GPIIb/IIa receptors) 
  • Factors that prevent secondary haemostasis
    • Antithrombin III antagonises the activity of thrombin
    • Protein C:
      • Activated by thrombin
      • Inhibits  thrombin,  Factor Va and Factor VIIIa
    • Protein S: co-factor for Protein C
    • Thrombomodulin:  co-factor for Protein C
  • Factors which promote fibrinolysis
    • Tissue plasminogen activator and urokinase activate plasminogen
    • Plasminogen is activated into p​​​​​​lasmin
    • Plasmin degrades fibrin into fibrin degradation products

An excellent, though somewhat dated, paper by the Rosenbergs (1984) can be recommended as the best peer-reviewed alternative to this unreliable online resource. It is still current enough for CICM First Part Exam purposes, as the expected answers to CICM questions will likely be based on what is written in popular med-school-level textbooks, and these have a tendency to reference each other, such that the lineage of every stated fact can be traced back to the first editions of the 1960s. In case one is not convinced, Yau et al (2016) is a more modern alternative which covers the topic to a satisfactory depth. Fibrinolysis is also done well by Chapin & Hajjar (2015) and Longstaff & Kolev (2015)

Factors that prevent the initiation of thrombosis

  • Normal endothelium has nothing thrombogenic on it. In order for coagulation to take place, a stimulating influence must be present, classically tissue factor. No tissue factor or collagen are present on the surface of the endothelium. 
  • Heparin-like proteoglycans are present on the surface of vascular endothelium. They are heparin like structurally, but also in the sense that in combination with antithrombin-III they have an anticoagulant effect. 
  • Glycocalyx has antithrombotic properties. For one it adsorbs all those aforementioned natural heparin-like molecules, as well as Antithrombin III. It also acts as a barrier, preventing thrombogenic basal lamina elements from coming into contact with the blood (Masola et al, 2021). 
  • Blood flow produces shear stress. This is a major mechanism which prevents blood from clotting, and - where a clot is forming - to prevent it from spreading, or becoming occlusive. Without paraphrasing too much of Hathcock (2006), it will suffice to say that enzymatic reactions (like the clotting cascade) behave by Michaelis–Menten kinetics in which the reaction rate depends on the concentration of substrate, and the constant movement of blood flow keeps taking activated enzymes and incompletely adherent platelets out of the area and off into some part of the circulation where they will be uselessly inactivated. 

Factors that limit clot propagation

Recalling the self-amplifying proteolytic cascade of haemostasis (such as the "thrombin burst" for example), the attentive reader will immediately realise that there must be some kind of regulatory process limiting this, because if there was not, the runaway sorceror's apprentice effect of this organic nanomachinery would result in the immediate and complete filling of the entire circulatory volume with clot. That this does not happen is a gratitude we owe to a number of naturally occurring factors that can be classified according to the part of the haemostatic process they are intended to oppose.

Factors that oppose platelet activity

  • Prostacyclin (PGI2) is released by the endothelium and inhibits platelet activation. The prostacyclin receptor is a Gs-protein coupled receptor on the surface of platelets that maintains platelets in a resting state 
  • Nitric oxide has a potent antiplatelet effect, which seems to be related to a combination of decreased intracellular calcium availability and the inhibition of thromboxane A2 receptors (Jin et al, 2005, do an excellent overview of this). Platelet activation is markedly impaired by these effects.
  • Fibrin degradation products act as inhibitors of platelet aggregation, because they readily bind to platelet GPIIb/IIa receptors, but then don't cross link with anything. This is one of the ways in which plasmin, a fibrinolytic agent, can inhibit platelet function (again this is from Jin et al).

Factors that oppose extrinsic pathway enzyme cascades

  • Tissue factor pathway inhibitor (TFPI) is an imaginatively named inhibitor of the tissue factor-FVIIa complex which forms on the exposed surfaces of broken blood vessels and other tissue planes (Broze, 1995). It is expressed on the surface of normal vascular endothelium, and opposes the initiation of the clotting cascade. This is probably one of the main mechanisms by which the early clot formation in primary haemostasis is limited to the injured surface only, i.e. only where normal endothelium is missing.

Factors that oppose intrinsic pathway enzyme cascades

  • Antithrombin III is a soluble mediator which acts as a protease inhibitor. Specifically, the protease it inhibits is thrombin itself. As thrombin is the main product of the early primary haemostatic pathway (activating platelets) and the dominant protease which forms the secondary haemostatic clot (by turning fibrinogen into fibrin), to oppose thrombin has a significant anticoagulant effect. Antithrombin III is the molecular drug target of heparin; the combination of ATIII and heparin has a significantly increased effect. Thrombin is also not the only thing that gets inhibited (many other clotting cascade factors are also reduced in activity, such as Factor IXa, Factor Xa, Factor XIa, and Factor XIIa). You may notice that many of these are factors involved in the amplification and propagation parts of the haemostatic process, managed by the "intrinsic pathway". Thus, antithrombin III activity can be measured by the APTT test.
  • Protein C,  which must be activated to Protein Ca, is a Vitamin K- dependent protease. Protein C is activated by thrombin itself, producing an excellent negative feedback phenomenon that automatically regulates excess thrombin activity. It also inhibits Factors FVa and FVIIIa with the help of Protein S as a co-factor. A recombinant version of active form was an attractive pharmacological agent to prevent DIC and organ dysfunction, but all attempts to turn it into money have failed.
  • Thrombomodulin is a co-factor for the thrombin-induced activation of protein C. It is expressed on the surface of endothelial cells. The appearance of thrombomodulin-thrombin complexes at the edges of a length of injured endothelium therefore limits the propagation of the clot.
  • Protein S is also a Vitamin K- dependent protease, and a co-factor for Protein C.

Natural processes of fibrinolysis

Question 19 from the second paper of 2015 asked complicated questions about fibrinolysis specifically and judging from the college examiners' notes, the expectations were high. The pass rate was a depressing 8%. This is not surprising, considering that there is a lot of material to cover. In short:

  • Fibrinolysis is the natural process of clot degradation which is mediated by circulating and endothelial proteolytic enzymes.
  • The main mediators of fibrinolysis are:
    • Plasmin is the most important endogenous thrombolytic actor. It is a serine protease, the activated form of plasminogen, and plasminogen is a circulating protein that gets activated by tissue plasminogen activator. So, really, you'd have to say that...
    • Tissue plasminogen activator (tPA) is the most important endogenous thrombolytic actor. This serine protease is found on all endothelial cells, and is responsible for maintaining a no-fibrin policy throughout the circulation. There is some basal level of fibrinolytic activity and some of this is also maintained by soluble hepatic tPA. Fibrin and plasminogen are required for its action, which makes logical sense.  Thus:
      • Plasminogen binds fibrin
      • tPA binds the plasminogen-fibrin complex and activates plasminogen
      • Activated plasminogen (plasmin) degrades fibrin into small peptide degradation product
    • Urokinase is another serine protease, also an activator of plasminogen, which is present on the endothelium. Unlike tPa, which is created by the endothelial cells themselves, urokinase is produced by monocytes, macrophages, and urinary epithelium. Its affinity for plasminogen is lower than that of tPa, but then it does not require fibrin as a cofactor.
  • Inhibitors of fibrinolytic pathways:
    • plasminogen activator inhibitor-1 and 2 (PAI-1,2): produced by the liver, inhibits tPA and urokinase
    • α2-antiplasmin: produced by the liver, a circulating serine protease inhibitor which inhibits plasmin directly by binding to it in a covalent manner
    • Thrombin Activatable Fibrinolysis Inhibitor (TAFI): a circulating inhibitor, activated by thrombin/thrombomodulin or plasmin; cleaves lysine residues on fibrin, preventing the binding of plasminogen to fibrin
  • Activators of fibrinolytic pathways:
    • Thrombomodulin binds to thrombin and activates Protein C
    • Protein C then inactivates TAFI and PAI-1/2

References

Rosenberg, R. D., and J. S. Rosenberg. "Natural anticoagulant mechanisms." The Journal of clinical investigation 74.1 (1984): 1-6.

Longstaff, Colin, and K. Kolev. "Basic mechanisms and regulation of fibrinolysis." Journal of Thrombosis and Haemostasis 13 (2015): S98-S105.

Yau, Jonathan W., Hwee Teoh, and Subodh Verma. "Endothelial cell control of thrombosis." BMC cardiovascular disorders 15.1 (2015): 1-11.

Ezihe-Ejiofor, J. Adanma, and Nevil Hutchinson. "Anticlotting mechanisms 1: physiology and pathology." Continuing Education in Anaesthesia, Critical Care & Pain 13.3 (2013): 87-92.

Ezihe-Ejiofor, J. Adanma, and Nevil Hutchinson. "Anticlotting mechanisms 2: pharmacology and clinical implications." Continuing Education in Anaesthesia, Critical Care & Pain 13.3 (2013): 93-97.

Broze Jr, George J. "Tissue factor pathway inhibitor." Thrombosis and haemostasis 74.07 (1995): 090-093.

Wood, Jeremy P., et al. "Biology of tissue factor pathway inhibitor." Blood, The Journal of the American Society of Hematology 123.19 (2014): 2934-2943.

Jin, Richard C., Barbara Voetsch, and Joseph Loscalzo. "Endogenous mechanisms of inhibition of platelet function." Microcirculation 12.3 (2005): 247-258.

Loscalzo, Joseph, and Eugene Braunwald. "Tissue plasminogen activator." New England Journal of Medicine 319.14 (1988): 925-931.

Jilani, Talha N., and Abdul H. Siddiqui. "Tissue plasminogen activator." StatPearls [Internet] (2021).

Chapin, John C., and Katherine A. Hajjar. "Fibrinolysis and the control of blood coagulation." Blood reviews 29.1 (2015): 17-24.

Masola, Valentina, et al. "Endothelial Glycocalyx as a Regulator of Fibrotic Processes." International Journal of Molecular Sciences 22.6 (2021): 2996.

Hathcock, James J. "Flow effects on coagulation and thrombosis." Arteriosclerosis, thrombosis, and vascular biology 26.8 (2006): 1729-1737.