Coagulation Flashcards
List 7 functions of thrombin
- Activates factors V, VIII, XI, XIII
- Activates platelets
- Inhibits fibrinolysis via thrombin-activatable fibrinolysis inhibitor
- Increases fibrinolysis via thrombin-thrombomodulin-EPCR pathway
- Generates fibrin from fibrinogen
- Mitogen
- Proinflammatory
- Stimulation of endothelin-1 production and release
- Inactivates ADAMTS13, leading to increased large vWF multimers
How does thrombin inhibit fibrinolysis?
- Thrombin-thrombomodulin complex activates Thrombin-Activated Fibrinolysis Inhibitor (TAFI)
- TAFI removes C-terminus from lysin residues on fibrin
- This reduces plasminogen and tPA (tissue plasminogen activator) binding, which reduces plasmin formation
- This mechanism is similar to tranexamic acid / aminocaproic acid (TXA/ECA)
How does thrombin increase fibrinolysis?
- Thrombin-thrombomodulin complex activates Protein C via the Endothelial Protein C receptor (EPCR)
- Indirect pathway: Activated protein C (aPC) inhibits FVa and FVIIIa, so less thrombin is formed, therefore less TAFI is formed
- Direct pathway: aPC inhibits plasminogen activator inhibitor 1 (PAI-1); therefore, tPA is uninhibited and can convert plasminogen into plasmin
- Protein S is an important cofactor for Protein C
Which pro-coagulation factors produced in the liver are vitamin K dependent?
2, 7, 9, 10
Anticoagulant rodenticides inhibit vitamin K epoxide reductase, which is required to replenish reduced vitamin K
Without reduced vitamin K, factors 2, 7, 9, 10 cannot be activated
Vitamin K1 should be used; do not use vitamin K3
Which anti-coagulation factors produced in the liver are vitamin K dependent?
Protein C and protein S
Comment on the use of each of these tests in a patient with anticoagulant rodenticide toxicosis, and state which is the preferred test:
PT
aPTT
PT & aPTT
VCM (TEG)
TAT
Fibrinogen levels
D-dimers
PVKAs
PT – this is the preferred test. Factor 7 has the shortest half-life in dogs, thus is depleted first (within 48 hours). PT requires Factor 7 activity.
aPTT – PTT does not test Factor 7 activity, though it will also become prolonged eventually; it is expected to be prolonged if clinical bleeding is related to anticoagulant rodenticide.
PT & aPTT – see above. In a clinically bleeding patient, both should be prolonged. For example, an haemothorax with a normal PT and/or aPTT is very unlikely due to rodenticide toxicity.
VCM (TEG) – The R-time/clotting time on viscoelastic testing corresponds to activating clotting time (ACT), i.e., represents secondary hemostasis. These tests can be used for evaluating anticoagulant rodenticide intoxicated patients. As in-house PT/aPTT testing becomes less common, the VCM by Entegrion is becoming a commonly used test for rodenticide toxicity.
Thrombin-Antithrombin complex (TAT) – Antithrombin binds thrombin to form TAT complexes, which are cleared by the liver. High circulating [TAT] implies increased thrombin generation, which would not be expected with anticoagulant rodenticide toxicosis (quite the opposite is expected).
Fibrinogen levels – this test would not be helpful. Fibrinogen is synthesised by the liver, thus is decreased in hepatic synthetic failure. Anticoagulant rodenticide toxicosis is a specific form of hepatic synthetic failure that is not related to global hepatic injury/dysfunction. Fibrinogen can be increased in inflammatory states.
D-dimers – this test would not be helpful. Plasmin cleaves cross-linked fibrin. The D-dimer is a specific antigen that is produced when fibrin crosslinked by Factor 13a is cleaved by plasmin. D-dimers represent a prothrombotic and/or hyperfibrinolytic state. “Proteolytic enzymes released by neutrophils can also degrade crosslinked fibrin exposing D-dimers” (eClinpath).
PIVKAs – in the absence of reduced vitamin K, inactive precursors of Factors 2, 7, 9, 10 can be detected and are known as “Proteins Induced by Vitamin K Absence.” The test is a modified PT test; needs to be sent to a lab, so takes significantly longer than PT, and has no obvious utility in veterinary medicine. In the olden days, PIVKAs were often reported as the academically ‘correct’ test for anticoagulant rodenticide toxicosis. Fortunately, we have become enlightened and can confidently tell old-school vets that PIVKAs serve no role in anticoagulant rodenticide management.
Describe the three major platelet stages that occur during primary hemostasis
- Adhesion/initiation:
When the endothelium is damaged, subendothelial components, such as von Willebrand’s Factor and collagen, are exposed.
Circulating platelets bind to these via specific receptors
GP1ba binds vWF
GPVI and a2b1 bind collagen
Binding via vWF is more important in high shear (arterioles and the microcirculation) states.
Binding collagen is more important in low shear (conducting vessels). - Activation/extension
Adherence leads to platelet activation, which includes shape changes to increase surface area; release of cytoplasmic dense granules and alpha granules; membrane flipping to provide a procoagulant surface; membrane metabolism to produce thromboxane A2 and proinflammatory lipids; activation of integrin a2bb3, which is the major fibrinogen receptor.
Local accumulation of platelet agonists (ADP, thrombin, thromboxane A2) recruits and activates additional platelets.
This leads to extension of the initial activated platelet monolayer. During activation, platelets change shape to become flatter and wider, so they cover a larger surface area and occupy a smaller volume; this allows formation of the initial platelet plug whilst allowing blood to continue to flow by (i.e., prevents occlusion).
*Aggregation/stabilization/perpetuation
Formation of the true, stable platelet plug. Adjacent activated platelets are firmly attached to each other via fibrinogen and the integrin a2bb3.
The clot retracts as it stabilizes and becomes resistant to dissolution.
Activated platelets release trophogens to promote endothelial repair.
List 5 drugs that inhibit platelet function, include MOA:
Platelet activation inhibitors
Aspirin**
Clopidogrel **
Congrelor
NSADIs *
Dipyridamole
Cilostazol
Prasugrel
Ticlopidine
Ticagrelor
Glycoprotein platelet inhibitors
Abciximab **
Eptifibatide*
Tirofiban
Protease-activate receptor-1 antagonists
Vorapaxar
What are the three types of von Willebrand’s disease?
vWD is an inhertited, autosomal, recessive disease.
· Type 1: decreased vWF with normal multimeric structure (i.e., all sizes of multimers are present and variably decreased).
o < 35% vWF:Ag concentration are at risk of haemorrhage.
o BMBT only becomes prolonged at < 20% vWF:Ag
o Bleeding is variable and often not a problem; it depends on the expression of the gene.
o Occurs in 60% of Dobermans – most hospitals have a Doberman policy. If someone wants to take the lead on developing this at the CVU, please let me know.
· Type 2: large multimers, which are the most effective for haemostasis, are absent. Absolute concentration of multimers may not be reduced.
o German short haired pointers
o Variable bleeding; can be severe.
· Type 3: marked reduction in concentration of all multimers.
o Dutch kooikers, Scottish terriers
o Variable bleeding; can be severe.
Explain the role of von Willebrand Factor in primary hemostasis
vWF is a glycoprotein multimer. There are large multimers, which are most important for haemostasis, and smaller multimers, which are progressively less effective in haemostasis. vWD Type 2 is a deficiency in the larger multimers.
vWF is synthesized by endothelial cells and stored Weibel-Palade bodies, and synthesized by platelets and stored in alpha-granules. It is released when the endothelium is damaged. There is also circulating vWF, which is released constitutively from the endothelium. Circulating vWF is bound to FVIII to prevent proteolysis of FVIII by protein C.
Damage to the endothelium causes vWF release. This happens to a greater degree in the arterial circulation due to activation by shear stress. Thrombin also causes release of vWF from the endothelium and platelets.
vWF from the endothelium remains anchored to the endothelial wall (by P-selectin) and binds to platelets via GP1b (glycoprotein 1b). Bigger vWF multimers have more binding sites, thus tether more platelets. vWF also binds strongly to subendothelial collagen (type IV).
In activated platelets, vWF also binds to integrin a2bb3. This binds platelets directly to the endothelium and, in high shear conditions (arterial thromboses) is thought to be responsible for platelet-platelet binding (aggregation); a2bb3 binding to fibrinogen is primarily responsible for platelet aggregation in low shear (venous) thromboses.
Is there a role for vasopressin in primary hemostasis?
Desmopressin is a synthetic analogue of antidiuretic hormone (ADH). It is increases renal permeability to water, resulting in lower volumes of highly concentrated urine and water retention. It has a much higher antidiuretic effect and much lower vasoconstrictor effect compared with vasopressin.
It causes the release of endothelial vWF via activation of the V2-receptor (the exact mechanism is unclear). Larger vWF multimers are preferentially released. FVIII release is also stimulated.
Desmopressin is first line in human Type 1 vWD for bleeding or prophylaxis before minor surgeries. Cryoprecipitate was also used previously, though has been progressively replaced by recombinant vWF. Desmopressin is also used in uraemic thrombocytopathy.
Limitations of desmopressin include tachyphylaxis and hyponatraemia (water retention). Desmopressin should be used cautiously in patients prone to thrombosis.
Vasopressin (and to a lesser degree desmopressin) activates platelets via V1a receptors.
What are microparticles and how might they contribute to hemostasis?
Microparticles are small, extracellular vesicles released by cells in response to activation or apoptosis. Platelet microparticles are thought to be the most abundant and are involved in coagulation.
Phosphatidylserine-positive microparticles provide a negatively charged surface for localization of coagulation factors and catalysation of coagulation reactions.
There is some evidence that anticoagulant microparticles also exist.
Microparticles can be produced by other cells. TF-bearing microparticles are a current field of research, as these microparticles are understandably procoagulant.
Should we recommend anti-thrombotic treatments for dogs with atrial fibrillation?
This is a tough question. Humans with atrial fibrillation are often anticoagulated. The recent CURATIVE statement summarized that dogs with cardiac disease are not at increased risk of thrombosis, based on the available data.
A dog with hypothyroidism, atrial fibrillation and arterial thrombosis is reported (Chow and French, JSAP, 2014). Atrial fibrillation resolved after treatment with levothyroxine.
Three dogs were reported with atrial fibrillation and thromboses in a different paper (Usechak et al., J Vet Cardiol, 2012). Two has left atrial thromboses identified via echocardiography.
Atrial fibrillation models have failed to result in thromboses in dogs (Nishida et al., Circulation: Arrhythmia and Electrophysiology, 2012).
A 2023 case report (Karn et al., JVIM) identified a right atrial thrombus that formed after cardioversion for atrial fibrillation and resolved with time/antithrombotic therapy.
Considering the available evidence, it seems reasonable to anticoagulate dogs with atrial fibrillation and with confirmed thromboembolic problems (e.g. arterial thrombosis, venous thrombosis or a thrombus seen on echocardiography). Dogs with atrial fibrillation and other factors contributing to a hypercoagulable state/thrombophilia are possible candidates for anticoagulation. Dogs with solely atrial fibrillation are unlikely to be candidates for anticoagulation.
ROTEM, TEG and VCM are all types of viscoelastic testing with similar graphical outputs. Label this diagram with the parameters for each of these tests.
What is the MOA for rivaroxaban?
On a VCM, where would it work primarily?
Rivaroxaban: Orally administered direct FXa inhibitor. Competitive inhibition of both free and clot bound FXa. Inhibits initial clotting time (CT, R, CT)
What is the MOA for tranexamic acid, and where on viscoelastic testing would it work primarily?
Tranexamic acid: a synthetic lysin analogue. Competitive and reversible inhibition of plasminogen binding sites, preventing conversion to plasmin. At higher concentrations, non-competitively inhibits plasmin. Inhibits fibrinolysis.
What is the MOA of clopidogrel, and where on VCM tracing would it work primarily?
Clopidogrel: irreversibly binds platelet P2Y12 receptors, preventing ADP binding-induced activation. Clopidogrel is a prodrug metabolized into active metabolites by the liver.
Inhibition of maximum amplitude (MCF, MA, MCF)
What is Scott syndrome?
Scott syndrome in humans is an autosomal recessive mutation resulting in an altered structure of the lipid scramblase enzyme. Consequently, phosphatidylersine is not externalized and the necessary procoagulant platelet surface is not formed. Reduced thrombosis and bleeding tendencies are seen.
Canine Scott Syndrome: is similar, but very rare. Seems to be a disorder of GSDs. Post-operative hemorrhage and epistaxis may occur
Ideally, flow cytometry is used to identify the lack of externalized phosphatidylserine
Explain heparin-induced thrombocytopaenia
A transient, immune-mediated drug reaction that occurs following exposure to heparin. It results in thrombocytopenia and concurrent thrombosis
Platelet Factor 4 (PF4) binds to heparin. IgG antibodies recognize heparin-bound PF4. Antibody binding causes a hypercoagulable state due to intravascular platelet activation and thrombin generation.
It occurs in <5% humans receiving unfractionated heparin (UFH) and <1% receiving low molecular weight heparin (LMWH). The 4Ts score which considered Thrombocytopenia, Timing, Thrombosis, and oTher (other) causes of thrombocytopenia is used to predict probability of heparin-induced thrombocytopenia prior to further testing. Diagnosis requires recent exposure to heparins, clinical signs, and confirmation of specific antibodies.
Treatment: 1) STOP all heparins, 2) anticoagulate with a different drug 3) investigate for thrombosis 4) do not give platelets to treat the thrombocytopenia. If thrombosis is present, human guidelines recommend 3 months of anticoagulation; without overt thrombosis, 3-4 weeks
Is there evidence for rebound hypercoagulability in veterinary medicine?
Rebound hypercoagulability is the rapid recurrence of a hypercoagulable state following withdrawal of anticoagulants. Presumably, this hypercoagulable state is more hypercoagulable than the pre-anticoagulated state. Rebound thrombosis would be the confirmed presence of thrombosis following anti-coagulant withdrawal.
Rebound hypercoagulability remains controversial in human medicine. Atrial fibrillation patients in which anticoagulants are abruptly discontinued seem to be at increased risk for thrombosis. The exact mechanisms are unclear: multiple may exist.
CURATIVE guidelines recommend that IV UFH be weaned to discontinuation and subcutaneous UFH weaned if possible. They do not recommend weaning LMWH. They suggest direct Xa inhibitors be weaned if possible.
