Haemostasis

Haemostasis is the consequence of a balanced interaction between blood cells, the vascular system, plasma proteins and low molecular weight substances. The body maintains the haemostatic system in continuous equilibrium in order that blood can circulate freely without alterations rendering it excessively dense (which would cause thrombosis) or fluid (which would cause haemorrhage).

PERFECT HAEMOSTASIS MEANS NO HAEMORRHAGE AND NO THROMBOSIS

For educational purposes, in order to understand the sequence of events, the process of haemostasis can be divided into:

• primary haemostasis
• secondary haemostasis
• tertiary haemostasis.

PRIMARY HAEMOSTASIS

Primary haemostasis comprises a vascular phase and a platelet phase. The former occurs following loss of vascular integrity, with exposure of subendothelial collagen, which leads to an immediate, but temporary, process of VASOCONSTRICTION (Fig. 1).

     Fig.1. Haemostasis: the vascular phase.

Vasoconstriction alters the normal laminar blood flow that characterizes the circulation of blood, creating turbulent flow. As a result of this turbulent flow, platelets can come into contact with subendothelial collagen, to which they adhere (PLATELET ADHESION PHASE) (Fig. 2).

     Fig. 2. Haemostasis: the platelet adhesion phase.

The adhesion is made possible by glycoprotein (GP) receptors on the platelet membrane. In particular, binding between von Willebrand’s factor, present in the endothelium in a filamentous form, and GP Ib/IX creates a bond between the subendothelium and platelets. Platelet adhesion is followed by a phase of platelet aggregation: once the platelets have adhered to the subendothelium they release chemical mediators (ADP and thromboxane) able to attract other platelets to the site of the vascular lesion in order to promote the formation of a platelet plug (Fig. 3). Specifically, it is GPIIb/IIIa that enables binding between single platelets.

      Fig. 3. Haemostasis: the platelet aggregation phase.

Although a platelet plug can become quite large, it is fairly unstable: restoration of normal blood flow would almost inevitably lead to removal of the thrombus, unless the plug is consolidated through the formation of fibrin, which is the key to secondary haemostasis (Fig. 4).

    Fig. 4. Secondary haemostasis.

SECONDARY HAEMOSTASIS

Secondary haemostasis is the process in which inactivated procoagulant proteins are activated, triggering a cascade whose terminal event is the production of insoluble fibrin that, like a net, stabilises the platelet plug permanently.

Traditionally the coagulation cascade is divided into intrinsic, extrinsic and common pathways (Fig. 5). The intrinsic pathway is activated by contact with subendothelial collagen and the proteins involved in the initial stage are kininogen, which complexes with prekallikrein, and factor XII (Hageman’s factor). The complex formation leads to activation of factor XII, which is transformed into an activated protease (factor XIIa) which, in its turn, catalyses the production of kallikrein from prekallikrein, with an increase in the production of factor XIIa (self-amplifying process) and of factor XIa, which then intervenes in the activation (protease formation) of other procoagulant proteins. The production of factor XIa then generates (in a cascade reaction) factor IXa, which activates and complexes factor VIII together with calcium ions (Ca_⁺²) and platelet phospholipids; at this point factor X (Stuart’s factor) is transformed into its active form (factor Xa) which gives rise to the common pathway.

The extrinsic pathway starts with factor VII (proconvertin) which, by complexing with tissue factor, a ubiquitous intracellular lipoprotein released following cell damage, and Ca_⁺²  activates the common pathway (factor X). The purpose of the common pathway, activated by both the intrinsic and extrinsic pathways, is to produce small amounts of thrombin, which generates insoluble fibrin after cleavage of fibrinopeptides A and B from the α and β chains of the fibrinogen molecule; subsequently the insoluble fibrin monomers undergo polymerisation and stabilisation, this latter helped by factor XIII (Laki-Lorand’s factor).

     Fig. 5. The coagulation cascade.

Given that coagulation is a self-amplifying process, in order to avoid the formation of an occlusive thrombus in a vessel and to enable removal of the clot once the tissue damage has been repaired, there must be a control mechanism for dissolving the thrombus (tertiary haemostasis).

TERTIARY HAEMOSTASIS

Tertiary haemostasis or fibrinolysis is activated by the production of a plasminogen-activator (t-PA) by the endothelial cells next to the lesion. This enzyme converts plasminogen into plasmin, a strongly proteolytic protein. Although plasmin is a non-selective protease, it acts mainly on the coagulum. Other enzymes with fibrinolytic activity are urinary plasminogen activator (u-PA) and streptokinase of bacterial origin.

CLINICAL APPROACH TO COAGULATION DISORDERS

Coagulation disorders can lead to haemorrhage and/or thrombosis. The clinician must be able to:

• recognize whether a given set of signs can be attributed to a coagulation disorder, be it acquired or congenital;
• understand which phase of the haemostatic process (primary, secondary or tertiary) is altered;
• choose the best diagnostic and therapeutic protocols to make the diagnosis and, when possible, achieve clinical resolution of the problem.

The history and clinical examination are useful for the first of the two above-mentioned points.

Blood loss in a young animal of certain breeds (for example, German Shepherd dog) should raise the suspicion of a congenital coagulation disorder (for example, haemophilia A). The intake of some drugs (for example, non-steroidal anti-inflammatory drugs, coumarins, heparin) could explain bleeding. Some disorders, such as pancreatitis, neoplasia, gastric torsion, heat stroke, and immune-mediated haemolytic anaemia, can be complicated by disseminated intravascular coagulation (DIC) (Fig 6).

Superficial bleeding (petechiae, ecchymoses, epistaxis) (Fig. 6) is a common indicator of altered primary haemostasis: in fact, with the activation of normally functioning secondary haemostasis, fibrin stops bleeding caused by qualitative and quantitative platelet disorders. Altered secondary haemostasis is characterized by bloody effusions in body cavities (for example, thorax, abdomen and joints) or large haematomas. In these cases the lack of fibrin production prevents the platelet plug from being stabilised, leading to severe haemorrhage.

Although superficial bleeding (epistaxis, haematemesis, haematuria and melaena) is more usually due to alterations in primary haemostasis, it is not uncommon to find it in cases of disturbed secondary haemostasis. With regards to thrombotic events, it is essential to understand whether these are the result of a generalised process (DIC) or a local event (for example, aortic thromboembolism due to feline cardiomyopathy).

There are various laboratory tests that can be of great help to the clinician in identifying a coagulation disorder (Table 1). Correct sampling, the choice of an appropriate anticoagulant and good storage of the sample are essential in order for these tests to be useful. A sample of blood with added K₃EDTA to make the fresh blood smear is sufficient for the platelet count and estimate, whereas for studies of platelet function and a satisfactory evaluation of secondary haemostasis, the blood must be collected into test-tubes containing sodium citrate (preferably buffered at 3.2%) as an anticoagulant in a 1:9 ratio with the blood.

INVESTIGATIONS OF PRIMARY HAEMOSTASIS

PLATELET COUNT AND ESTIMATE
It is essential to know the platelet count (determined instrumentally) and platelet estimate (determined by microscopic evaluation) in order to understand alterations in primary haemostasis. The microscopic evaluation is used to check whether the instrumentally measured platelet count is realistic and to determine platelet morphology: for example, the presence of numerous platelet aggregates can lead to an artificially low platelet count, in that an aggregate can be erroneously counted as a single platelet by an automatic platelet counter; contrariwise, the presence of marked lipaemia can lead to a falsely high platelet count.

Changes in the platelet count/estimate can indicate the presence of thrombocytopenia or thrombocytosis.

There are four different mechanisms underlying thrombocytopenia:

• increased consumption of platelets (DIC)
• destruction of platelets (immune-mediated thromobocytopenia)·lack of production of platelets (bone marrow failure)
• sequestration of platelets (organomegaly, haemorrhage).

In the presence of thrombocytopenia clinical signs (for example, petechiae, Fig. 6) usually appear when the platelet count/estimate is below 30,000 cells/µl. The same clinical signs may occur in qualitative platelet disorders, in which the platelet count may be normal, but the platelets do not function correctly: a defect of von Willebrand’s factor or a state of hyperviscosity (hypergammaglobulinaemia or marked erythrocytosis) can explain a defect in platelet adhesion. In contrast, the use of anti-aggregant drugs can interfere with platelet aggregation.

The common finding of thrombocytopenia in the  Cavalier King Charles Spaniel (Fig. 7) is worth noting; an asymptomatic macrothrombocytopenia is observed frequently in dogs of this breed. This abnormality is due to an inherited genetic mutation in beta1-tubulin, which affects the formation of pro-platelets from megakaryocytes. The consequence is a low number of platelets which appear particularly large (giant platelets) (Fig. 8).

Thrombocytosis, on the other hand, can be caused by:

• inflammation (reactive thrombocytosis)
• corticosteroids
• iron-deficiency anaemia
• primary chronic myeloproliferative disorders affecting only the megakaryocyte lineage (essential thrombocythaemia) or in association with concomitant myeloid and erythroid hyperplasia (polycythaemia vera)
• splenectomy
• rebound thrombocytosis (following immunosuppressive treatment for an immune-mediated thrombocytopenia).

Marked thrombocytosis can cause thrombosis.

The platelet estimate is a procedure that can be performed in emergencies, in the absence of an automatic cell counter: it is considered that one platelet in an oil-immersion field (100x) corresponds to approximately 15,000 platelets/µl (Fig. 9).

BUCCAL MUCOSAL BLEEDING TEST
This test can exclude defects in primary haemostasis in a subject that does not have clinical signs of bleeding on examination. The animal is placed in lateral decubitus and the upper lip is folded upwards and held up with a gauze to prevent venous return. Using a specific lancing device, two incisions are made in the mucosa. Excess blood around the incision can be blotted away with absorbent paper, but the incision site itself should not be touched in order not to remove any clot. The buccal mucosal bleeding time (BMBT) corresponds to the time passed from the incision to when the bleeding stops. A BMBT of about 3 minutes is considered normal, whereas a bleeding time longer than 5 minutes is considered abnormally prolonged. Given the poor standardisation of this test, the results should be interpreted with care.

BONE MARROW STUDIES

Cytological examination of the bone marrow in cases of thrombocytopenia is used mainly to determine the number and morphology of cells of the megakaryocyte lineage (Fig. 10). In the case of thrombocytopenia the presence of megakaryocytic hyperplasia could suggest an immune-mediated thrombocytopenia and/or myelodysplasia. Absence or paucity of megakaryocytic lineage cells could be due to bone marrow aplasia/hypoplasia or to a neoplastic proliferative disorder in the bone marrow with consequent myelophthisis (compression and replacement of the resident bone marrow cells by the neoplastic population).

SEARCH FOR ANTI-PLATELET ANTIBODIES
Flow cytometry can reveal antibodies (IgG/IgM) that may be present on the surface of platelets in cases of immune-mediated thrombocytopenia. The investigation should be carried out on unrefrigerated whole blood preserved with sodium citrate, within 24-36 hours of collection of the blood.

THE PLATELET FUNCTION ANALYSER-100^®
The platelet function analyser-100^® (PFA-100^®) enables a fast evaluation of defects of primary haemostasis, with particular focus on defects of platelet adhesion (for example, von Willebrand’s factor deficiency). The investigation should be carried out on a sample of whole blood to which sodium citrate has been added. The test should be performed within a few hours (at most, 4 hours) of collecting the blood.

VON WILLEBRAND’S FACTOR
Both antigen tests and genetic investigations are available to study deficiencies of von Willebrand’s factor. Levels of von Willebrand’s factor antigen (vWF:Ag) are determined using an enzyme-linked immunosorbent assay (ELISA) on plasma obtained from whole blood with sodium citrate additive centrifuged at 3000 rpm for 10 minutes (or at 4500 rpm for 5 minutes).

• The genetic investigations enable identification of homozygous or heterozygous mutations in some breeds predisposed to von Willebrand’s factor deficiency.·type I von Willebrand’s disease: tests in the Dobermann, Bernese, Coton de Tulear, Pinscher, Kerry Blue Terrier, Manchester Terrier, Pembroke Welsh Corgi, Miniature Poodle, Scottish Sheepdog
• type II von Willebrand’s disease: tests in the German Shorthaired Pointer, German Wirehaired Pointer 
• type III von Willebrand’s disease: tests in the Scottish Terrier and Shetland Sheepdog.

Whole blood in K₃EDTA or two buccal swabs vigorously rubbed against the gums and collars of teeth are needed to perform these genetic tests.

INVESTIGATIONS OF SECONDARY HAEMOSTASIS

ACTIVATED CLOTTING TIME
The aim of the activated clotting time (ACT) test is to evaluate the intrinsic and common pathways of the coagulation cascade in a rapid and simple manner; a diatom (alga) is used for this purpose, since this activates the intrinsic pathway with consequent clotting of the blood. Two millilitres of blood are introduced into a diatom-coated test-tube which is kept at a steady temperature of 37°C and gently and repeatedly turned upside down until clotting occurs. The time from when the blood is introduced into the test-tube to when the clot forms is measured. A normal value of the ACT in a dog is between 90-120 seconds, while that in the cat is < 75 seconds. The pathological conditions considered able to prolong the ACT are DIC, liver disorders, poisoning by vitamin K antagonists and haemophilia A and B. However, the poor sensitivity of the test severely limits its use. The results of the test may be abnormal in the case of severe platelet function defects or severe thrombocytopenia even in the absence of alterations of secondary haemostasis.

PROTHROMBIN TIME
The purpose of this test is to evaluate the extrinsic and common pathways of the coagulation cascade. The clotting proteins must be very substantially reduced in order for the prothrombin time (PT) to increase, given that the concentrations of some factors are particularly high in the dog (factors VII and V). This means that even a slight lengthening of the PT indicates a severe coagulation problem.

Some conditions that commonly cause prolongation of the PT are:

• DIC
• poisoning by rodenticides
• liver failure
• primary hyperfibrinogenolysis
• congenital deficiencies of clotting factors in the extrinsic pathway (for example, FVII deficiency in Beagles)
• nephrotic syndrome
• a high haematocrit (excess anticoagulant with respect to the plasma)
• incorrect sampling or error in the ratio of blood to anticoagulant.

Shortening of the PT, although fairly rare, can be seen in states of hypercoagulability.

ACTIVATED PARTIAL THROMBOPLASTIN TIME
The purpose of measuring the activated partial thromboplastin time (aPTT) is to evaluate the intrinsic and common pathways of the coagulation cascade. Some conditions that commonly cause prolongation of the a PTT are:

• DIC
• poisoning by rodenticides
• liver failure
• primary hyperfibrinogenolysis
• congenital deficiencies of clotting factors in the intrinsic pathway (for example, haemophilia A: factor VIII deficiency)
• nephrotic syndrome
• a high haematocrit (excess anticoagulant with respect to the plasma)
• incorrect sampling or error in the ratio of blood to anticoagulant.

Shortening of the aPTT, although fairly uncommon, can be seen in states of hypercoagulability.

FIBRINOGEN
Fibrinogen, a positively modified acute phase protein, may increase during inflammatory states and thereby mask possible coagulation disorders caused by its consumption. Corticosteroids, pregnancy and oestrogens can also increase the plasma concentrations of this protein.

The levels of fibrinogen may be reduced in patients with :

• DIC
• liver failure
• hereditary hypofibrinogenaemia (Saint Bernard, Borzoi)
• primary hyperfibrinogenolysis
• malabsorption
• effusions into body cavities.

FIBRIN AND FIBRINOGEN DEGRADATION PRODUCTS
Fibrin and fibrinogen degradation products (FDP) are produced by the proteolytic effect of plasmin on fibrin and, to a lesser extent, on fibrinogen.

An increase in FDP suggests:

• DIC
• liver failure
• primary hyperfibrinogenolysis
• immunosuppressive therapy
• bleeding into body cavities.

D DIMERS
D dimers are the products of degradation of fibrin, but not of fibrinogen. Events that can cause a rise in D dimers are:

• DIC
• thrombosis.

ANTITHROMBIN
Antithrombin is a circulating anticoagulant with an inhibitory effect on thrombin. A decrease in the activity of antithrombin can be caused by excessive consumption of the protein (DIC), by a decrease or absence of its production (for example, liver diseases) or by excessive loss (for example, during an enteropathy).

THROMBOELASTOGRAPHY
Thromboelastography has been recently introduced into veterinary medicine. This test enables a real-time evaluation of the phase of platelet aggregation and defects of secondary haemostasis. These latter include DIC, hypercoagulability, hyperfibrinogenolysis and congenital or acquired deficiency of coagulation factors. A limitation of thromboelastography is that it must be performed on whole blood with sodium citrate additive within 30 minutes of taking the blood sample.

Tab. 1. Summary of the laboratory changes in common coagulation disorders.

N= normal; D= decreased; I= increased; PFA-100^®= platelet function analyser-100^® ; PT= prothrombin time; aPTT= activated partial thromboplastin time; FDP= fibrin and fibrinogen degradation products; AT= antithrombin.

Suggested readings

1. Davis B., Toivio-Kinnucan M., Schuller S. et al. – Mutation in b1-Tubulin Correlates with Macrothrombocytopenia in Cavalier King Charles Spaniels – J Vet Intern Med 2008;22:540–545
2. Feldman B.F., Zinkl J.G., Jain N.C. – Schalm’s Veterinary haematology, 5° edition, ed Lippincott Wlliams & Wilkins, 2000
3. Pedersen H.D., Haggstrom J., Olsen LH., et al. – Idiopathic asymptomatic thrombocytopenia in Cavalier King Charles Spaniels is an autosomal recessive trait – J Vet Intern Med 2002; 16:169-173
4. Stockham S.L., Scott M.A. – Fundamentals of Veterinary Clinical Pathology – 1° edition, Blackwell 2002
5. Willard M.D., Tveden H. – Small Animal Clinical Diagnosis by laboratory Methods – 4° edition, Saunders 2004