Introduction

Definition: Primary hemostasis is defined as the formation of the primary platelet plug. This
serves to plug off small injuries especially in microvessels (< 100 m) in mucosal tissues
(respiratory, gastrointestinal, genitorurinary tracts). Platelets are not only involved in platelet plug
formation but are also crucial for formation of fibrin (secondary hemostasis). Activated platelets
express a negatively charged phospholipid, phosphatidylserine (PS), on their surfaces, which is a
binding site for the assembly of coagulation factor complexes. Assembly of these complexes on
this surface markedly amplifies fibrin formation (called the propagation phase of secondary
hemostasis).
Constituents: Platelets, von Willebrand factor (vWf), and the vessel wall.

Cells: Endothelial cells (source of vWf), platelets

Adhesive proteins: vWf, subendothelial matrix protein (e.g. collagen)

Facilitators: Thrombin (platelet agonist)

Primary hemostasis

Sequence of events: Normally, the intact endothelium is a physical barrier separating circulating
platelets from thrombogenic substances (such as extracellular matrix proteins) in the
extravascular space. When the endothelium is injured, the procoagulant subendothelial matrix
(consisting of proteins such as collagen, laminin, and fibronectin) is exposed and immediately
initiates primary hemostasis, which consists of three main events:

Platelet adhesion: Platelets adhere to the exposed subendothelial matrix (directly or

indirectly via vWf).

Platelet activation: Once platelets adhere, they then become activated and recruit (and
activate) additional platelets to the injured site. Also, thrombin generated by the coagulation
cascade is an extremely powerful platelet activator.

Platelet plug formation: Fibrinogen forms bridges between activated platelets to form
the platelet plug.

More detail on these three events is given below.

Note: Secondary hemostasis (formation of fibrin by coagulation factors) is usually initiated
simultaneously (through exposure of tissue factor on subendothelial fibroblasts). Activated
platelets promote fibrin formation and provide a physical scaffold on which fibrin formation
proceeds.

Sequence of events

Platelet adhesion
Upon endothelial injury, platelets bind to exposed subendothelial matrix proteins with the aid of
adhesion molecules or receptors on their surfaces. These receptors are transmembrane
glycoproteins (GP) and are either integrins or non-integrin receptors. Platelets adhere to exposed
collagen in the subendothelial matrix when cell surface receptors (that are constitutively
expressed) encounter exposed thrombogenic subendothelial matrix proteins. There are three
main receptors that mediate platelet adhesion:
1.

GPVI and 21 (an integrin): These bind directly to collagen.

2.

GP1b-IX-V: This binds to vWf, which acts as a bridge or glue between extracellular
matrix proteins and the receptor complex of on the platelet surface. vWf is produced by
endothelial cells (where it is stored in Weibel-Palade bodies) and is secreted into the
subendothelial matrix and plasma (thus it can be measured in plasma). GPIb-IX-V/vWf
interactions are important for mediating platelet adhesion to vessels with high shear rates.
vWf is actually a multimeric molecule that is quite large. Studies have shown that under high
shear, the molecule unfurls and can act as a sling to capture platelets streaming by the injury.
Abnormalities in either GP1b-IX-V complex or vWf can result in a hemorrhagic diathesis.
These are usually inherited disorders known as Bernard-Soulier syndrome (GP1b deficiency)
or von Willebrand disease (vWD; abnormality or deficiency in vWf). Of these conditions, only
vWD has been reported in animals.

Platelet activation
The adhesion of platelets to subendothelial matrix proteins activates the platelets (increasing
intracellular calcium and inducing cell signaling) and causes a variety of changes in platelets:

Shape change: Platelets change from their normal discoid shape to elongated cells with
cytoplasmic extensions, which increases their surface area.

The release reaction (degranulation): Platelets release the contents of preformed

cytoplasmic granules (-granules and dense or -bodies).
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-granules contain vWf and coagulation factors, including fibrinogen, Factor V

(FV) and FXIII. Once released, these factors can participate in platelet plug formation
(vWf, fibrinogen) or fibrin formation (FV, FXIII). Of these factors, only vWf is produced in
megakaryocytes; the remaining proteins are endocytosed from plasma. There are also
species differences in the amount of vWf within platelets, with dog platelets having little
vWf, whereas cat platelets can contain up to 20% vWf. These granules also contain
adhesion molecules, such as P-selectin, which is expressed on the platelet surface after
activation. Detection of P selectin is actually used as an in vitro marker of platelet
activation in the laboratory. In vivo, P selectin binds to its ligand, PSGL-1, on endothelial
cells and leukocytes. This helps firmly platelets bind to the endothelium and
allows leukocytes to be incorporated into developing clots (leukocytes, like platelets, also
provide a surface scaffold for fibrin formation).

Dense granules are rich in ADP and serotonin, both of which are platelet agonists
(i.e. they recruit and activate additional platelets). They store calcium which is needed
for secondary hemostasis and platelet activation. Recent studies have shown that they
are also stores for short-chain polyphosphate (chains of phosphate molecules). These
polyphosphates have been shown to have multiple roles in hemostasis and are both
procoagulant (they promote the cofactor activity of Factor V in the common pathway and
also serve as a cofactor for thrombin activation of FXI in the amplification phase of
secondary hemostasis) and antifibrinolytic, i.e. they help form dense fibrin fibrils which
are more resistant to fibrinolysis. Dense granule secretion can be measured by detecting
the amount of 14C-serotonin or ATP released during aggregation.

Phospholipid metabolism (membrane metabolism): Activation of phospholipase

A2 hydrolyzes membrane phospholipids (phosphatidylcholine) to arachadonic acid. This is the
initial substrate for subsequent reactions involving several enzymes (e.g. cyclo-oxygenase,
COX-1) which produce eicosanoids, particularly thromboxane A2, which are powerful platelet
agonists. Pharmacologic inhibition of COX-1 can be accomplished by aspirin and nonsteroidal anti-inflammatory agents. These inhibitors are used to prevent thrombotic events in
both people and animals (principally dogs).

Phosphatidylserine exposure (membrane flipping): The normal platelet membrane is

asymmetrical with the more negatively charged phospholipid, phosphatidylserine (PS), being
preferentially and actively maintained on the inner membrane leaflet. Phosphatidylserine is
very important, because it is the membrane surface on which the coagulation cascade
occurs, i.e. it is the binding site for coagulation factors and actually amplifies their activity,
thus promoting fibrin formation. When platelets are activated, PS is flipped to the outside of
the platelet membrane, allowing coagulation factors to bind to the activated platelet.
Phosphatidylserine used to be called platelet factor 3 (PF3). An inherited defect in PS
exposure (Scott syndrome) has been reported in German Shepherd dogs and results in
excessive hemorrhage in affected dogs.

Release of procoagulant microparticles (microvesiculation): Activated platelets shed

tiny microparticles, that are enriched in PS, from their membrane surfaces. These
microparticles extend the surface area of the platelet, thus providing a larger surface on
which fibrin formation can proceed. Dogs with Scott syndrome also have a defect in
microvesiculation. Phosphatidylserine exposure and microvesiculation can be assessed in
the laboratory with flow cytometry.

Recruitment of additional platelets (outside-in signaling): Additional platelets are

recruited to the site of vessel injury by agonists that are released by activated platelets
themselves (ADP, thromboxane A2) or that are formed from activated coagulation factors
(thrombin). These agonists bind to surface receptors on platelets and trigger outside-in
signaling which activates the recruited platelets. This outside-in signaling involves G proteins
and other facilitator molecules, such as guanine exchange factors (GEF). An important GEF
is CalDAG-GEF1 (calcium diacylglycerol-regulated guanine exchange factor-1) which is

downstream of the ADP receptor and has shown to be defective in Basset Hounds and
Eskimo Spitz with thrombopathia. Important platelet receptors for agonists are:
P2Y12: This is the receptor for ADP. Clopidogrel is a P2Y12 antagonist that is also

used as an antithrombotic drugs in humans, dogs and cats. Efficacy in horses is less
certain.
Protease-activated receptors (PAR): They are unique G-protein coupled

receptors expressed on several different cells. They carry their own ligand, which is only
expressed after the receptor is cleaved to expose the ligand, which then activates the
receptor. Once activated, the receptor cannot be re-activated even though the cleavage
enzyme dissociates from the receptor. These receptors bind several agonists, but the
main agonist is thrombin, which binds to PAR-1 and PAR-4 expressed on human
platelets, with PAR-1 being the main activator.

Activation of the fibrinogen receptor (inside-out signaling): An integrin, IIb3, also

known as GPIIb/IIIa or CD41/61, is the fibrinogen receptor on platelets. It is normally present
on platelet surfaces (indeed it is a platelet-specific marker) but cannot bind fibrinogen on
unstimulated platelets. Once platelets are activated, the fibrinogen receptor changes
conformation on the outside of the platelet due to intracellular signaling (inside-out), which
allows fibrinogen to bind adjacent platelets, forming platelet aggregates. Binding of fibrinogen
to activated platelets can be used as a laboratory marker of platelet activation. A genetic
defect in GPIIb/IIIa has been detected in various breeds of dogs (Otterhound, Great
Pyrenees) and cattle. This results in a bleeding disorder called Glanzmanns thrombasthenia.
A protein called kindlin is required for correct functioning of integrins in platelets and there
has been a report of a dog with kindlin deficiency. The dog had combined leukocytosis, due
to a leukocyte adhesion deficiency (integrins are required for leukocytes to migrate into
tissue), and hemorrhagic defect due to defective platelet aggregation. Platelet aggregation
can be analyzed using platelet agonists in aggregometers. Platelet adhesion and aggregation
in response to agonists can also be evaluated using platelet function analyzers (PFA).

Platelet aggregation
This is mediated primarily by fibrinogen, which binds to the activated fibrinogen receptor
(GPIIb/IIIa) on platelets. This links platelets together forming the primary plug. The platelet plug is
sufficient to stop bleeding from most small blood vessels in response to every day trauma (e.g.
eating) or venipuncture, however in larger vessels or with more severe damage, the primary
platelet plug must be stabilized to cease the hemorrhage. This is accomplished through
secondary hemostasis or fibrin production by coagulation factors. Also note, that platelets have
roles beyond what has been described for hemostasis. For instance, it is now known that
platelets are essential for vascular integrity. It is possible that hemorrhage in patients with
thrombocytopenia is not only due to an absence of platelets but also to a loss of vascular
integrity.

Inhibitors

There are physiologic, pathologic and pharmacologic inhibitors of primary hemostasis.

Physiologic: Endothelial cells release prostacyclin (PGE12) and nitric oxide, both of
which inhibit platelet activation. They also form a physical barrier, preventing platelets from
being exposed to thrombogenic subendothelial matrix proteins.

Pathologic: 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).

Pharmacologic: These inhibit the following platelet activation events. Note: Drugs that
inhibit platelet function can precipitate or worsen hemorrhage in an animal with a mild
hemostatic disorder.
Arachidonic acid metabolism: Aspirin (irreversibly) and non-steroidal anti-

inflammatory drugs (reversibly) inhibit cyclo-oxgenase, which is needed for thromboxane

A2 production.
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ADP agonist activity: Clopidogrel is an ADP receptor antagonist.

Clinical signs

Epistaxis in a Bassett with inherited thrombopathia(photograph courtesy of Dr. Marjory Brooks)

A defect in any aspect of primary hemostasis can manifest as hemorrhage, typically from
mucosal surfaces (the sites where the primary platelet plug is important due to rapid fibrinolysis).
Hyperactivity in primary hemostatic components is a cause of hypercoagulability and a risk factor
for thrombosis.

Defective primary hemostasis: Platelet plug formation is inadequate with a defect in

any primary hemostasis event, including thrombocytopenia. This usually manifests as
hemorrhage from small blood vessels subjected to daily minor trauma, i.e. those supplying
mucuosa (e.g. epistaxis, hematuria). Hemorrhage can be spontaneous with severe defects or
induced by trauma or surgery with milder defects. Since platelets plug small holes in vessels,
thrombocytopenia or abnormal platelet function (thrombopathia) can result in pinpoint
mucosal or cutaneous hemorrhages (petechiae). Petechiae are not typical in vWD. Bruises
(ecchymoses) can also occur.

Hyperactive primary hemostasis: Platelet hyper-reactivity and increased vWf

concentrations contribute to hypercoagulability, i.e. are risk factors for thrombosis, but are
unlikely to induce thrombosis alone. Thrombocytosis in animals is not usually associated with
abnormal thrombotic events.

Sample collection
Two samples are required to evaluate primary hemostasis:

EDTA-anticoagulated blood: Platelet count, genetic testing (if needed).

Citrate-anticoagulated plasma: All other tests.

Please refer to the sample collection page for additional guidelines on how to collect samples
appropriately to optimize hemostasis test results.

Tests
It is imperative that a platelet count is done in all animals presenting with clinical signs of
excessive hemorrhage. Platelet counts can be quantified using automated analyzers or can be
semi-quantified (estimated) from a well-prepared fresh blood smear. Additional platelet testing
requires fresh blood or, preferably, referral of the patient to a testing center that is capable of
performing advanced hemostasis testing. Few tests of primary hemostasis are available to
general practitioners. These include a platelet count, a buccal mucosal bleeding time (BMBT),
genetic testing for specific inherited disorders, and measurement of vWf concentrations.

Screening tests: Platelet count, BMBT, platelet function analyzer testing.

Specific tests: von Willebrand factor antigen (vWf:Ag), genetic testing for vWD and
defined inherited thrombopathias (e.g. CalDAG-GEF1 defect in Bassett Hounds and Eskimo
Spitz, GPIIb/IIIa deficiency or Glanzmans thrombasthaenia in Otterhounds and Great
Pyrenees)

Specialized testing: Flow cytometric tests for platelet activation (P selectin expression),
function in primary hemostasis (platelet aggregation, release reaction), procoagulant activity
(PS exteriorization, microparticle quantification, thrombin generation assays), plateletassociated antibodies, reticulated platelets.

Disorders

Defects arise in any aspect of the hemostatic pathway. As indicated above, these defects can
result in deficient hemostasis (hypocoagulability), which can result in excessive hemorrhage, or
accelerated hemostasis (hypercoagulability), which can result in thrombosis. Disorders can also
be inherited or acquired. Inherited conditions should be suspected in young animals presenting
with episodes of recurrent hemorrhage or thrombosis. Acquired disorders are more likely in older
animals with underlying disease. The most common inherited defect in primary hemostasis is von
Willebrand Disease and thrombocytopenia is the most common acquired defect (there are
multiple causes of thrombocytopenia).