Inhibitors

Physiologic inhibitors

There are physiologic inhibitors for every stage of hemostasis. Some of these inhibitors are physical barriers, e.g. the endothelial cell acts as a physical obstruction between platelets and the matrix proteins that bind and activate them (von Willebrand factor and collagen) and between coagulation factors and their cofactors and substrate, specifically factor VII (even though tiny amounts circulate in active form) is separated from its cofactor, tissue factor, and its substrate, factor X, by an intact endothelium. Once that endothelial barrier is disrupted, all bets are off and platelets stick and aggregate forming a platelet plug (primary hemostasis), they express phosphatidylserine and support thrombin generation, which is initiated by factor VII binding to TF expressed constitutively on fibroblasts just below the endothelial cells (secondary hemostasis). A summary of physiologic inhibitors of hemostasis is given below.

Physiologic inhibitors serve essential functions in hemostasis. They serve to maintain the balance between procoagulant (formation of a platelet plug or a fibrin clot) and fibrinolytic (lysis of the clot). This makes sense, because we do not want the clot lysing as it is being formed (see hemostatic pendulum). They also restrict normal hemostasis to the site of vessel injury, preventing it from disseminating throughout the vasculature. When this inhibition is overwhelmed (e.g. excessive activation of hemostasis or a “hypercoagulable” state) or inhibitors are dysfunctional or deficient, thrombosis or hemorrhage can result. Thrombosis will occur if inhibitors that normally restrict fibrin formation or platelet function are deficient. Hemorrhage will occur if there is a deficiency of inhibitors that usually restrict fibrinolysis. In animals, inherited deficiencies of inhibitors are rare and most are acquired defects (secondary to underlying disease, particularly disseminated intravascular coagulation, the prototypical thrombotic disorder).

Pathologic inhibitors

There are also pathologic inhibitors or inhibitors that arise because of an underlying disease state. Known inhibitors of hemostasis are high concentrations of fibrin(ogen) degradation products and high concentrations of monoclonal immunoglobulins (e.g. multiple myeloma).  We can also cause inhibitor formation by our actions (which are usually necessary). For instance, treatment of dogs with severe factor VIII deficiency (hemophilia A) can result in the generation of antibodies to factor VIII, making the dog refractory to future transfusions. Inhibitory antibodies against phospholipids can also be seen in immune-mediated diseases, e.g. systemic lupus erythematosis.

• Primary hemostasis: High concentrations of fibrin(ogen) degradation products can inhibit platelet aggregation (fragments D and E have a high affinity for platelet membranes and compete with fibrinogen for platelet receptors, thus impairing aggregation) as can paraproteins (high concentrations of monoclonal immunoglobulins with multiple myeloma – the monoclonal protein coats platelets, interfering with platelet aggregation, adhesion and phospholipid exposure).
• Secondary hemostasis: High concentrations of FDPs and monoclonal immunoglobulins can inhibit fibrin formation (specifically fibrin polymerization). Inhibitors of coagulation factors can arise in certain autoimmune diseases, e.g. antiphospholipid antibodies in systemic lupus erythematosis or with treatment of animals with severe inherited factor deficiencies (see above).
• Fibrinolysis: Polyphosphates have been shown to inhibit fibrinolysis (they make the fibrin clot more dense, which is lysis-resistant). Polyphosphates are found in dense granules of platelets and are released on platelet activation. They are also found in bacteria and are longer in size than those found in platelets. Polyphosphates associated with bacterial sepsis may inhibit fibrinolysis, promoting thrombosis in affected patients.

Pharmacologic inhibitors

Naturally, sometimes we deliberately administer drugs to inhibit hemostasis. For example, to prevent thrombosis in immune-mediated hemolytic anemia (IMHA) in dogs and cardiomyopathy in cats, animals are often treated with aspirin (which inhibit COX1 in platelets), clopidogrel (blocks the ADP receptor on platelets) or heparin (which inhibits multiple coagulation factors in secondary hemostasis promoting the action of antithrombin). We also take advantage of heparin’s anticoagulant action to use it as an anticoagulant to collect blood samples for chemistry testing. Naturally, we cannot reverse this effect of heparin so samples anticoagulated with heparin should not be used for screening coagulation assays (e.g. prothrombin time, activated partial thromboplastin time).

• Primary hemostasis: 
  • Aspirin (irreversibly) and non-steroidal anti-inflammatory drugs (reversibly) inhibit cyclo-oxgenase, which is needed for thromboxane A2 production. Aspirin also inhibits prostacyclin production in endothelial cells (inhibits platelet function), but higher doses are required for this undesirable affect than those used to inhibit COX in platelets. Importantly, the inhibitory effect of aspirin persists for the lifespan of the platelet (about 6 days in the dog), therefore simple drug withdrawal will not aid platelet function (have to wait until new platelets are produced..
  • Clopidogrel is an ADP receptor antagonist. After metabolism, the drug inhibits the P2Y₁₂ receptor for ADP, preventing ADP from activating platelets.
• Secondary hemostasis:
  • Heparin: This is frequently administered as an anticoagulant in animals with hypercoagulability (e.g. immune-mediated hemolytic anemia in dogs). Both fractionated and unfractionated heparin act as anticoagulants via antithrombin. The activity of heparin depends on the number of pentasaccharides. More pentasaccharides as found in unfractionated heparin inhibit both FXa and thrombin. In contrast, shorter chain heparin as found in low molecular weight or fractionated heparin only inhibit FXa. The latter have more predictable pharmacodynamics and are less likely to cause excessive bleeding. The inhibitory effect of heparin can be evaluated by prolongation of the APTT (unfractionated) or decreased FXa activity (unfractionated and low molecular weight heparin). The latter is more commonly used to monitor heparin therapy.
  • Warfarin: This inhibits recycling of vitamin K, resulting in a relative deficiency of vitamin K-dependent factors (FII, VII, IX, X). Warfarin is seldom used to inhibit secondary hemostasis therapeutically in animals due to a low therapeutic:toxicity ratio (animals bleed severely when on this drug).
• Fibrinolysis: Tranexamic acid is synthetic lysine. Since plasminogen (and plasmin) bind to lysine residues in fibrin and fibrin promotes the activity of  tissue plasminogen activator (tPA), lysine acts as a competitive inhibitor for plasmin and plasminogen, inhibiting fibrinolysis. Lysine analogues also stimulate the release of α2-antiplasmin from endothelial cells. ε-aminocaproic acid works similarly (it is an analogue of lysine) but is less potent than tranexamic acid in humans. Both of these drugs inhibit fibrinolysis (as assessed by a tPA-induced fibrinolytic assay in vitro using viscoelastic clot detection) in horses, although horses may require 20 fold lower concentrations of the drugs than humans (based on the tPA-induced fibrinolytic assay, which showed that lower concentrations of the drug were required to inhibit tPA-induced fibrinolysis of fibrin formed in plasma of healthy horses than inhibition of fibrinolysis of fibrin formed in human plasma) (Fletcher et al 2013). The drugs are mostly used to treat hemoperitoneum in horses (e.g. ruptured uterine artery) (Arnold et al 2008), although there are no well-controlled studies showing the drugs are efficacious for this purpose.

Inhibitor summary

Below is a table summarizing endogenous (physiologic) inhibitors of hemostasis, separated by process.

Physiologic inhibitors	Mechanism of action
General inhibitors
Endothelium	Global hemostasis: Physical barrier; Primary hemostasis: Secrete platelet antagonists, e.g. prostacyclin, nitric oxide, ADPase; Secondary hemostasis: Express thrombomodulin, a receptor which binds thrombin and then activates protein C (inhibits the cofactors; FVa, FVIIIa), and the endothelial protein C receptor (cofactor for protein C activation). Express surface heparin-like glycosaminoglycans (GAGs), which Enhance AT and TFPI activity; Fibrinolysis: Secrete fibrinolytic inhibitors, e.g. plasminogen activator inhibitor-1.
Plasma proteins	Global hemostasis: Several, e.g. α2-macroglobulin, α1-antitrypsin. These are non-specific inhibition of proteases (activated coagulation factor enzymes, plasmin etc). These are produced in the liver.
Secondary hemostasis
Tissue factor pathway inhibitor (TFPI)	Inhibits the tissue factor-factor VIIa (TF-FVIIa) and TF-FVIIa-FXa complexes. Its activity enhanced by heparin-like GAGs and protein S. Rapidly inhibits any free factor Xa generated by the TF-FVIIa complex on fibroblasts during the initiation phase of secondary hemostasis. Produced in endothelial cells.
Antithrombin (AT)	Inhibits multiple coagulation actors, including thrombin, FXa, FXIIa, FXIa, FIXa, TF-FVIIa. Activity is enhanced by heparin (exogenous) and heparin-like GAGs on endothelial cells. Produced in the liver.
Protein C	Inactivates FVa, FVIIIa. This is activated by thrombin (which downregulates its own production) by thrombin binding to a receptor on endothelial cells, thrombomodulin. There is also an endothelial protein C receptor, which amplifies the activation of protein C by thrombomodulin-thrombin complex and also stimulates anti-inflammatory effects. Vitamin K dependent and produced in liver.
Protein S	Cofactor for TFPI; Cofactor for activated protein C. Protein S is found free and bound to an acute phase protein, C4-binding protein. Only free protein S is able to act as a cofactor for TFPI and activated protein C. Vitamin K dependent and produced in the liver.
Plasmin	Inactivates the cross-linker, FXIIIa (Hur et al 2015)
Pathologic inhibitors	FDPs and monoclonal immunoglobulins can interfere with fibrin polymerization; antibodies can develop against coagulation factors in some immune-mediated diseases (e.g. systemic lupus erythematosis)
Fibrinolysis
Thrombin-activatable fibrinolysis inhibitor (carboxypeptidase B)	Binds to lysine residues in fibrin, preventing plasminogen binding and activation; activated by the thrombin burst (amplification and propagation phases of secondary hemostasis). Produced in the liver.
Plasminogen activator inhibitor-1 (PAI-1) and -2 (PAI-2)	Binds and inactivates tissue plasminogen activator. Produced by endothelial cells and inhibited by thrombin-thrombomodulin complex. PAI-1 is thought to be the main in vivo inhibitor.
Antiplasmin	Binds and inactivates plasmin. Produced in the liver.
Polyphosphates	Form a dense fibrin network, which resists lysis. Released from dense granules in platelets during activation (release reaction).
Extracellular nuclear material (histones, DNA, high mobility box proteins)	Binds to the fibrin network, inhibiting fibrin degradation, promotes PAI-1 inhibition of tPA (Gould et al 2015)