Diagnosis of coagulopathies

Question 1

Primary haemostasis (simple)

Answer 1

The formation of a platelet plug

Question 2

Secondary haemostasis (simple)

Answer 2

Activation of coagulation factors to result in the formation of cross-linked fibrin which binds to and stabilises the platelet plug

Question 3

Primary haemostasis - formation of the platelet plug (detailed)

Answer 3

Initiating event in haemostasis

Blood vessel injury -> vasoconstriction -> subendothelial matrix exposed -> platelets stick to this layer with Von Wilebrand factor acting as a bridge

Once adhered the platelets activate - change shape, up-regulate fibrinogen receptors, release contents of granules (Attracts more platelets)

Temporary repair of vessel but weak and easily washed away

Activation also exposes negatively charged phospholipid that provides link to secondary haemostasis

Question 4

Secondary haemostasis (detail)

Answer 4

Coagulation cascade (ezymatic reactions) and results in formation of fibrin to stabilise platelet plug

The coagulation cascade is divided into the intrinsic pathway, the extrinsic pathway and the final common pathway.

Activation of first factor leads to second etc., positive feedback loops accelerate clotting once begun

Achored to the phospholipid so localises to site of injury

The ultimate aim of secondary haemostasis is the production of thrombin (factor IIa) which catalyses the conversion of fibrinogen (factor I) to fibrin (factor Ia).

Calcium needed at several points
Factors produced in the liver (severe liver disease can cause lack of factors)

Question 5

Which factors are vitamin K dependent

Answer 5

Factors II, VII, IX, and X

They are produced as inactive precursors and activated by Vitamin K. This results in formation of Vitamin K epoxide, which must be reduced back to Vitamin K to allow continued use and factor activation (rodenticides interfere with this).

Question 6

Extrinsic/tissue factor pathway

Answer 6

Release of tissue factor from damaged tissue or activated endothelial cells.

Binds factor VII and in the presence of calcium activates it leading to initiation of the extrinsic pathway and common pathway and ultimately the formation of thrombin.

The small amount of thrombin generated activates platelets and factor XII, therefore resulting in additional activation of the intrinsic pathway.

The thrombin also activates factors V and VIII to provide feedback activation.

Question 7

Intrinsic pathway

Answer 7

Amplifies the response and leads to accelerated coagulation.

NB certain ‘contact factors’ will also result in activation of the intrinsic pathway and this phenomenon is utilised in certain clotting tests and explains why blood clots when it contacts glass or plastic tubes.

The contact pathway is important for coagulation tests in vitro, is less important for in vivo haemostasis (many mammals with mutations leading to inactive factor XII do not have bleeding tendencies), but the contact factors do have physiological roles in inflammation and may play a role in pathological thrombus formation.

Question 8

Common pathway

Answer 8

Leads to the production of thrombin (II) which catalyses the conversion of fibrinogen to fibrin.

Thrombin also activates factors V, VII and IX to further promote coagulation, and protein C and protein S to inhibit coagulation.

Finally, thrombin activates factor XIII which catalyses the formation of cross linked fibrin to stabilise the clot.

Question 9

Control of haemostasis

Answer 9

There are naturally occurring inhibitors for every stage of haemostasis, which limit the extent of thrombus formation and/or initiate fibrinolysis.

The most important factor is anti-thrombin (III) which inhibits thrombin.

Protein C and Protein S (both vitamin K dependent) are also important inhibitors as is tissue factor pathway inhibitor.

Excessive thrombosis is usually the consequence of insufficient inhibition.

Question 10

Fibronolysis

Answer 10

Process of breaking down fibrin, which is facilitated by plasmin.

Tissue plasminogen activator converts plasminogen to plasmin, which then cleaves fibrinogen and fibrin so breaking down thrombi.

When fibrinogen and fibrin are broken down fibrin degradation products (FDPs) are formed.

When fibrin is crosslinked a new antigen is created.

When crosslinked fibrin (i.e. part of a stabilised thrombus) is degraded this new antigen is revealed on the fragments and can be detected (D-dimers).

Thrombin activates fibrinolysis (via activation of protein C and protein S) in order to balance clot formation with clot dissolution and limit the extent of thrombus formation.

Question 11

Categories of blood clotting disorders

Answer 11

Haemorrhagic disorders
- disorders of primary haemostasis
- disorders of secondary haemostasis
- hyperfibrinolytic disorders

Thrombotic disorders

Question 12

Disorders of primary haemostasis

Answer 12

Thrombocytopaenia - insufficient platslets

Thrombocytopathia - platelets present but not functioning correctly

Lack of vWF

Question 13

Disorders of secondary haemostasis

Answer 13

Acquired deficiencies of factors e.g. marked hepatocellular dysfunction, rodenticide toxicity, DIC

Inherited deficiencies of coagulation factors e.g. Haemophilia A

Question 14

Thrombotic disorders

Answer 14

Blood stasis

Decreased activity of anti-coagulant factors (protein-losing enteropathy)

Decreased or impaired fibrinolysis

Increased endogenous procoagulants

Question 15

Tools for diagnosis of haemostatic disorders

Answer 15

Signalment and clinical history

Clinical exam findings

Screening lab tests (CBC, biochem)

Coagulation tests

Genetic testing and specific tests for coagulation factors

Question 16

Clinical history for haemostatic disorders

Answer 16

Signalment – age, breed, sex

Details of bleeding – site, severity, frequency, age at first episode, family, type (petechiae vs. intra-cavitatory, history, initiating factors e.g. trauma, surgery vs. spontaneous)

History of access to toxins e.g. rodenticides, oestrogens

Drug history e.g. NSAIDs

Travel history - infectious diseases

Question 17

Clinical signs of disorders of primary haemostasis

Answer 17

Only a small amount of blood leaks out before a fibrin clot seals the injury so tend to see small haemorrhages – petechiae and ecchymoses (larger than petechiae) in the skin, mucous membranes and sclera.
Purpura are confluent petechiae.

May get bleeding from mucosal surfaces e.g. from the gastro-intestinal tract (leading to melaena), epistaxis (uni- or bi-lateral) or from the urinary tract (haematuria). Occasionally CNS bleeding may occur with diverse signs related to CNS dysfunction.

Excessive bleeding may occur after surgery or trauma.

NB petechiae not commonly seen in vWD

Question 18

Causes of primary haemostatic disease

Answer 18

Marked thrombocytopaenia (most common) - signs when <50x0^9/L

Lack of vWF

Thrombocytopathia

Question 19

Tests for primary haemostasis

Answer 19

Platelet count

Bleeding time - BMBT (buccal mucosal bleeding time), (cuticle bleeding time)

Von Willebrands factor

Question 20

Buccal mucosal bleeding time

Answer 20

This is a test of primary haemostasis but is not specific for any particular cause.

Carry out if the platelet count is normal (expecting there to be a problem with clotting factors etc).

A superficial cut is made in the gum that is shallow enough to be sealed with a platelet plug only and not require fibrin formation.

A simplate (spring loaded device) is used to produce a standardised cut in the mucosa of a dog that is restrained with the lip tied back with gauze.

Timing should commence once the cuts are made.

Excess blood should be absorbed with filter paper from adjacent to the cuts (do not dab the cut or the platelet plug may be disturbed) every 5-10 seconds.

Stop timing once the plug is formed and bleeding stops.

Normal bleeding times in healthy dogs are <3.3 mins but may be mildly prolonged (<4.2) in sedated or anaesthetised dogs.

Healthy anaesthetised cats BMBT are <3.3 mins.

If bleeding persists for 10 minutes + stop the test and apply pressure.

Question 21

What can cause a prolonged BMBT?

Answer 21

THrombocytopaenia - so check BEFORE

Thrombocytopathia

vWd - screen Dobermans pre-surgery, if prolonged delay elective surgery and test

Question 22

Diagnosis of von Willebrands disease

Answer 22

Prolonged BMBT

vWF assay

Genetic test

Question 23

Thrombocytopathia

Answer 23

Inherited or acquired.

Some inherited thrombocytopathias exist such as Chediak Higashi syndrome in Persian cats.

Acquired are most common and include:
* Hyperglobulinaemia
* Neoplasia
* DIC
* NSAIDs – as a result of COX inhibition (usually not causative of spontaneous bleeding
* Severe renal or hepatic disease
* Essential thrombocythaemia

Question 24

Von Willebrands disease

Answer 24

Lack of vWF (produced by endothelial cells and megakaryocytes in response to endothelial damage)

vWF attached to subendothelium, binds to platelets to anchor them to injury and each other - platelet plug

When activated by thrombin it triggers activation of coagulation casacade

Common in Dobermans, rare in cats

Question 25

Clinical signs of von Willebrands disease

Answer 25

Severity of signs if variable and heterozygotes may not show clinical signs until surgery. Clinical signs are those of a primary haemostatic disorder, although petechiation is uncommon for reasons that are not currently understood.

Question 26

Diagnosis of von Willebrands disease

Answer 26

* Normal platelet count * Buccal mucosal bleeding time prolonged * Poor clot retraction * vWF assay * Genetic testing Measure von Willebrand antigen using an ELISA. The results are expressed as a percentage of a control group of dogs Values >70% are considered normal. Values <50% are consistent with vWd. Values between this are equivocal and may be normal (but carriers) or may be affected. With low values there is a risk of bleeding. Although BMBT may be prolonged in vWd it is too insensitive to use for diagnosis, it will only be prolonged if vWf antigen is <20%. There are also tests for specific gene mutations in certain breeds. These are useful for breeding programmes but do not tell you how much vWf antigen is present which is useful to assess potential risk of bleeding.

Question 27

Treatment of von WIllebrands disease

Answer 27

Palliative only Prophylactiv treatment in dogs with vWD prior to surgery (cryoprecipitate, fresh frozen plasm, fresh whole blood) Desmopressin can be used as is cuases release of vWF from endothelial cells - response unpredictable but can increase vWF levels for 4 hrs

Question 28

Clinical signs of disorders of secondary haemostasis

Answer 28

Defects in the coagulation cascade usually result in more severe bleeding. The platelet plug forms but it is unstable since not stabilised by the fibrin network and blood flow can wash it away resulting in bleeding. Bleeding may occur into body cavities e.g. joints or subcutaneous tissue with bruising developing (but rarely petechiae). Haematomas may form with excessive haemorrhage following surgery, trauma or venepuncture. Mucosal or surface bleeds are less common. Delayed bleeding or re-bleeding may occur.

Question 29

Tests to assess secondary haemostasis

Answer 29

* Whole blood clotting time (WBCT) * Activated clotting time (ACT) * Activated partial thromboplastin tine (APTT) * One stage prothrombin time (OPST aka PT) Ensure sample handled correctly, use citrate tube

Question 30

Whole blood clotting time

Answer 30

Crude screening test. Blood is drawn into a plain glass tube - Contact with the glass triggers (negative charge) the intrinsic pathway. The time taken for a clot to form is recorded. The tube is tilted every 30 seconds to examine for the presence of a clot. A normal result is < 6 minutes. This test assesses the intrinsic and common pathways but is very insensitive. It will only be prolonged if there is a severe factor deficiency (<5% normal). It is also affected by a range of other variables e.g. temperature, size of tube, PCV, thrombocytopaenia (due to a deficiency of platelet phospholipids required for the clotting cascade). Therefore may be used in an emergency but is not advised for routine work.

Question 31

Activated clotting time

Answer 31

This is a modification of the WBCT. Evaluates the intrinsic and common pathways. The tube contains diatomaceous earth, which is a chemical activator of the intrinsic pathway. The test is more standardised as it is performed at 37oC in pre-warmed tubes. Again the time for a clot to form following addition of blood to the tube is timed. This test is more sensitive than the WBCT but is still only prolonged if there is severe deficiency of the factors (<10%). It also relies upon platelet-derived phospholipids and is also prolonged with severe thrombocytopenia.

Question 32

Activated partial thomboplastin time (APTT)

Answer 32

Evaluates the intrinsic and common pathways and is performed on plasma. A contact activator (e.g. kaolin) of the intrinsic pathway is added to the plasma. An exogenous source of phospholipids and calcium are provided and the test is performed under controlled conditions. So apart from the clotting factors all of the ‘ingredients’ of the coagulation cascade are provided and the test is not affected by platelet count. This test is more sensitive, it will be prolonged if the coagulation factors are <30% of their normal level.

Question 33

One stage prothrombin time (OSPT or PT)

Answer 33

Tests the extrinsic common pathway and is performed on citrated plasma. Tissue factor is provided as an activator for the extrinsic pathway and as with the APTT, calcium and a source of phospholipids are included. Factor VII has the shortest half-life and so OSPT is sensitive to factor VII deficiency (and so potentially with rodenticide poisoning as factor VII has the shortest half life and is depleted first therefore the OSPT will become prolonged before the APTT).

Question 34

Abnormal APTT and OSPT times

Answer 34

If more than 20-25% of the control

Question 35

Causes of disorder of secondary haemostasis (acquired coagulopathies)

Answer 35

Hepatic disease ○ Check bile acids, urea, albumin, bilirubin. Vitamin K antagonism (rodenticide toxicity) ○ Antagonism (rodenticide toxicity). ○ Deficiency (GI or biliary disease, diet, EPI). Circulating inhibitors of clotting cascade (e.g. heparin from mast cell tumours) Antibodies to clotting factors Consumption of coagulation factors. DIC, Angiostrongylus vasorum.

Question 36

Vitamin K antagonism

Answer 36

Factors II, VII, IX and X are produced by the liver in an inactive form and must be converted to the active, carboxylated form by the action of vitamin K. This reaction leaves vitamin K in its inactive epoxide form and it must be converted back to the active form of vitamin K by epoxide reductase. Many common rodenticides act on epoxide reductase and other reductases to inhibit this reaction and stop the recycling of vitamin K. Absorption of vitamin K from intestines cannot keep up with demand for vitamin K, therefore rapid depletion of the active form of vitamin K and hence activated factors II, VII, IX, and X also become rapidly depleted. Factor VII has the shortest half-life (6 hours) and hence becomes depleted first. Therefore, deficiencies in the extrinsic coagulation pathway develop initially, and so the OSPT will become prolonged first in this sort of toxicity.

Question 37

Diagnosis of rodenticide toxicity

Answer 37

History Clinical signs - signs of secondary haemostatic disorders) - dyspnoea due to haemothorax or pulmonary bleeding - abdominal bleeds - haematomas Haematology - anaemia if significant blood loss - platelet count mildly decreased in severe bleeds Biochem - rule out liver disease Imaging - to define sites of haemorrhage Coagulation testing - OSPT prolonged first, good indicator of toxicity - ultimately OSPT, APTT, WBCT, and ACT all prologned PIVKAs can be sensitive markers of toxicity but not widely available Some labs to tox screens for it

Question 38

Hepatic disease and haemostatic disorder

Answer 38

Clotting factors are produced by the liver, and severe diffuse hepatic disease can result in depletion of clotting factors and increased bleeding tendency. Prolonged biliary obstruction can also reduce vitamin K absorption. When considering hepatic biopsy, coagulation times should be checked and vitamin K supplemented if coagulation times are prolonged. If still prolonged despite vitamin K therapy then fresh plasma could be given prior to liver biopsy.

Question 39

Disseminated intravascular coagulation (DIC)

Answer 39

Balance of coagulation and fibrinolysis is perturbed leading to widespread coagulation and subsequent exhaustion of the normal fibrinolytic process. Marked inflammatory diseases (e.g. acute pancreatitis) will trigger endothelial damage, platelet activation and release of tissue procoagulants. This leads to widespread activation of clotting cascades and formation of clots which will lodge and block small blood vessels, leading to reduced perfusion, vascular compromise and end organ damage (hypercoagulable phase of DIC). This widespread activation leads to consumption of platelets and coagulation factors. Activation of clotting also leads to activation of fibrinolysis, which consequentially inactivates clotting factors and breaks down existing clots, which in turn releases fibrin degradation products (FDPs) which inhibit coagulation further. Widespread activation of fibrinolysis also leads to depletion of anticoagulant factors such as antithrombin II, protein C and protein S (hypocoagulable or haemorrhagic phase of DIC).

Question 40

Clinical signs of DIC

Answer 40

Clinical signs in severe DIC may be related to end organ damage (renal damage, CNS damage). Haemorrhage may also occur due to depletion of platelets and coagulation factors and activation of fibrinolysis. In less severe DIC, the clinical signs can be subtle or absent.

Question 41

Diagnosis of DIC

Answer 41

* Inflammatory leukogram * Thrombocytopenia * Prolonged OSPT/APTT * Elevated D-dimers/FDPs * Low plasma fibrinogen concentrations Other supportive features would include biochemical evidence of end organ damage and the presence of red cell fragmentation features such as schistocytes and acanthocytes.

Question 42

Treatment of DIC

Answer 42

* Treat the underlying cause if possible * Maintain organ perfusion * Support renal function with dopamine +/- frusemide if necessary * Oxygenate to reduce hypoxia * Heparin (low dose) can be given to help reduce intravascular coagulation * Plasma/whole blood may be needed to replace clotting factors Prognosis can be poor if the underlying cause is not addressed.

Question 43

Angiostrongylus vasorum (lungworm)

Answer 43

Canine parasite that causes primary and secondary coagulopathies in the UK Larvae in slugs and snails - dog eats Migrate from gut to end arterioles in lungs, mature, lay eggs, larvae - coughed up and swallowed Causes DIC Prolonged OSPT/APTT, thrombocytopaenia, and elevated D-dimers

Question 44

Diagnosis of Angiostrongylus vasorum

Answer 44

SNAP test (ELISA) on blood Faecal flotation to see larvae Sometimes larvae seen in BAL sample

Question 45

Treatment for Angiostrongylus vasorum

Answer 45

Imidocloprid + moxidectin spot on (Advocate) or fenbendazole Anti-inflammatory doses of preds can be used to avoid pneumonitis assoicated with parasite death

Question 46

Inhertied coagulopathies (secondary haemostasis)

Answer 46

Haemophilia A

Question 47

Haemophilia A

Answer 47

Inherited defect, especially German Shepherd Dogs. Associated with a deficiency in factor VIII. Males are more commonly affected than females. The severity of signs is variable, with those with most severe disease showing spontaneous bleeding into body cavities and less affected dogs only showing clinical signs following surgery.

Question 48

Diagnosis of Haemophilia A

Answer 48

Supported by finding an increased APTT with normal OSPT, WBCT, and ACT are also prolonged Definitive diagnosis achieved by measurement of factor VIII

Question 49

Treatment of Haemophilia A

Answer 49

No definitive treatment available and owners often request euthanasia once diagnosis is confirmed Treatment will aim to supply factor VIII, ideally by giving cryoprecipitate, however more commonly fresh frozen plasma or whole blood are used The problem is that transfusion reactions may develop if blood or plasma are used

Question 50

How to assess disorders of fibrinolysis

Answer 50

Fibrin degredation products D-dimer Thromboelastography

Question 51

Fibrin degredation products

Answer 51

Plasmin cleaves fibrinogen and fibrin to form degradation products that can be detected with agglutination based assays. FDPs are low in normal animals and will be increased in states of increased fibrinolysis e.g. disseminated intravascular coagulopathy and other thromboembolic diseases such as iliac thrombosis in cats and internal haemorrhage. FDPs are cleared by the liver and kidneys, therefore renal dysfunction or liver disease could contribute to high FDPs.

Question 52

D-dimer

Answer 52

A specific type of degradation product from fibrin that has been crosslinked – i.e. that has come from a clot and not just fibrinogen. D-dimers are also detected in agglutination assays. D-dimers are low in healthy animals. High values >2000mg/ml indicate thromboembolic disease (i.e. DIC or a thrombus) but lower values (1000-2000) are non-specific and may occur in a range of conditions e.g. generalised inflammation, neoplasia, thromboembolic disease, or internal bleeding. D-dimers will also be increased in renal or hepatic dysfunction.

Question 53

Thromboelastography

Answer 53

Can only be assessed on freshly drawn blood samples - within 4hrs Citrated whole blood used, placed in a cuvette. A sensor is inserted into the sample and detects when a clot forms between the sensor and the side of the sample cup and how long it takes to reach a certain size Output provides a global view of clotting - can also detect hypercoagulable states as well as hypocoagulable states

Question 54

Causes of thrombosis

Answer 54

Blood stasis – e.g. cats with HCM, which can lead to aortic thromboembolism Decreased activity of anticoagulant factors – e.g. PLN (loss of ATIII in urine) Decreased or impaired fibrinolysis – e.g. hyperadrenocorticism, because steroids inhibit fibrinolysis Increased endogenous procoagulants – e.g. IMHA

Question 55

Sequelae of thrombosis

Answer 55

Thrombi can tend to lodge in the pulmonary vessels which can lead to pulmonary thromboembolism (PTE), severe respiratory compromise, pulmonary hypertension and death.

Question 56

Treatment of thrombosis

Answer 56

may involve aspirin and heparin but is controversial.