Platelets: Formation, Function & Morphology

Platelets

• Platelets are small, sometimes overlooked blood cells critical for hemostasis.
• They are cytoplasmic fragments without a nucleus in mammalian species.
• Platelets originate from megakaryocytes, large bone marrow cells with lobulated nuclei.
• Megakaryocytes undergo endomitosis, replicating DNA and cytoplasm without dividing into separate cells.
• The lifespan of platelets in circulation is about 5 to 10 days, with species variation.
• Platelets are either utilized or phagocytized in the liver when they reach the end of their lifespan, or if they done get used up before then.
• Desialylation of the platelet membrane triggers phagocytosis in the liver.
• The liver produces thrombopoietin, which stimulates megakaryocyte proliferation and platelet release.
• Platelets have sialic acid on their membrane, which are recognized by receptors in the liver by Ashwell-Morrell receptors as they age and lose the sialic acid.
• Platelets provide primary hemostasis by sealing vascular defects.
• Platelets adhere to the subendothelial extracellular matrix at the site of vascular damage, which helps to activate the clotting cascade.
• Platelets typically have a light blue cytoplasm with magenta granules and are smaller than erythrocytes.
• Activation during sample collection or smear preparation can cause platelets to display cytoplasmic projections.
• Platelets contain granules with substances that activate other platelets, release coagulation factors, regulate vascular tone, and promote tissue healing.
• Activated platelets aggregate to form a platelet plug, leading to the formation of a platelet thrombus.
• Platelet activation involves reactions between substrates and receptors.
• Collagen binding in the extracellular matrix, ADP, thromboxane A2, and serotonin activate platelets.
• Endothelium secretes prostacyclin to inhibit excessive platelet activation.
• Platelet activation releases calcium, leading to downstream effects.
• Phospholipases are activated, freeing arachidonic acid from the cell membrane.
• Arachidonic acid is converted into thromboxane A2 via the COX-1 pathway to activate more platelets.
• Platelets change shape, increasing surface area to seal vascular defects and provide a surface for the coagulation cascade.
• Activated GPIIb/IIIa receptors on the platelet membrane bind fibrinogen.

Alpha Granules

• These are visible using light microscopy as magenta granules.
• Alpha granule contents include platelet factor 4, which neutralizes heparin and attracts monocytes, fibrinogen, coagulation factors V, VII, XI, XIII, P-selectin, plasminogen activator inhibitor, and tissue factor pathway inhibitor.

Dense Granules

• Dense granules require electron microscopy for visualization.
• Dense granule contents include nucleotides like ADP, ATP, GTP, GDP, Ca2+, Mg2+, serotonin (which induces vasoconstriction), and histamine (which causes vasodilation).

Thromboxane A2 (TXA2)

• Synthesized by activated platelets from arachidonic acid metabolism via cyclooxygenase-1 and thromboxane synthetase.
• Thromboxane A2 promotes vasoconstriction, platelet activation via a positive feedback loop, and platelet aggregation through GPIIb/IIIa expression.

GPIIb/IIIa

• This integrin αIIbβ3 is found on the platelet membrane and within alpha granules.
• GPIIb/IIIa is normally inactive until activated, upon which it undergoes a conformational change.
• GPIIb/IIIa binds to fibrinogen, von Willebrand factor (VWF), and fibronectin.
• GPIIb/IIIa facilitates platelet binding to each other and to the subendothelial matrix.
• GPIIb/IIIa can bind to other GPIIb/IIIa receptors by linking fibrinogen or VWF.

Von Willebrand Factor

• Also known as "Factor VIII-related antigen."
• VWF is a glycoprotein that forms large multimers, where high-molecular-weight multimers are most effective for platelet adhesion.
• Found in platelets (α-granules), endothelial cells (Weibel-Palade bodies), and circulation.
• VWF functions in platelet adhesion by forming a bridge between platelets and collagen via the GpIb receptor and by binding to clotting factor VIII, increasing its half-life.
• It allows platelets to lock into place and onto each other.

Platelet Regeneration

• Increase of Thrombopoietin leads to increased platelet production.
• Increased numbers of megakaryocytes in the bone marrow is observed.
• Platelets tend to be larger (increased MPV) and have more variation in size.
• Large platelets can also arise from other causes.
• Platelet size can be used to assess if the bone marrow is working, but it is not definitive.

Feline Platelets

• In felines, platelets normally vary widely in size.
• Feline platelets have a strong tendency to clump in blood tubes, which can falsely decrease the platelet count.

Non-Mammalian Thrombocytes

• Thrombocytes are functional equivalents of platelets.
• Thrombocytes retain nuclei.
• Thrombocytes are typically the smallest blood cells.
• Thrombocytes have basophilic nuclei with clear or light-blue cytoplasm that often forms blebs.
• These cells sometimes have small granules.

Primary Hemostasis Defects: Clinical Manifestations

• Minor manifestations include:
  • Skin issues such as petechiae and ecchymoses
  • Mucosal bleeding such as epistaxis, hematuria, and melena
  • Excessive post-operative bleeding

Platelet Assessment

• Platelet numbers/mass can be assessed with:
  • Manual platelet count
  • Automated platelet count/MPV/Pct
  • Quantitative buffy coat
  • Slide estimate
• Platelet function tests include:
  • Buccal Mucosal Bleeding Time (BMBT)
  • Thromboelastography (TEG)
  • Platelet Function Analyzer (PFA-100)

Manual Platelet Count

• The hemocytometer is used for this procedure.
• Sample dilution and RBC lysis are required.
• The manual platelet count is considered the "gold standard" but has poor precision.
• Manual methods have poor precision due to the small blood volume and potential for human error.

Automated Platelet Count: Impedance

• Cells disrupt an electrical current and create a voltage peak.
• Peak height corresponds to cell size such that large cells are RBCs, and small cells are platelets.
• Large platelets may be interpreted as RBCs, causing a spuriously decreased platelet count.
• Small RBCs may be counted as platelets, causing a spuriously increased platelet count.

Automated Platelet Count: Laser

• Cells pass through a laser beam, creating light scatter that depends on size and granularity.
• This method allows for better separation of RBCs and platelets compared to impedance methods.
• Large platelets and small RBCs can still cause spurious results.
• Lipid droplets/other particles may be counted as platelets.

Mean Platelet Volume (MPV)

• MPV is the average volume of all platelets, similar to MCV for RBCs.
• MPV may increase in patients with increased platelet production, but decreased MPV is insignificant.
• Possible MPV errors include those caused by platelet clumping, particulates such as lipid droplets or cell fragments, or analyzer exclusion of large platelets.

Quantitative Buffy Coat (QBC)

• Buffy coat area is expanded with a plastic "float" so that WBC and platelet layers are stained.
• Platelets are expressed as % of blood volume (plateletcrit or Pct).
• This process is the best assessment of total platelet mass in hemostasis and regulation of thrombopoietin.
• This device is not as useful for WBC assessments.
• Platelet mass matters more than platelet count.

Automated Analyzer: Calculated Plateletcrit

• (Platelet count x MPV)/1000, equivalent to the % of volume, and similar to hematocrit.
• Some values can be erroneous, such as spuriously decreased platelet count (clumping), and errors in MPV.

Platelet Estimation

• Estimating the platelet numbers from blood smears can be determined by averaging the number of platelets per 100x field in a monolayer of the smear.
• The platelet estimate can be found by multiplying the average by 15,000 to 20,000.
• The crude platelet estimate is imprecise and is affected by clumping.
• Always perform to verify thrombocytopenia as significant clumping indicated it's likely spurious.

Platelet Function Testing

• Some tests include:
  • Buccal Mucosal Bleeding Time (BMBT)
  • Thromboelastography (TEG)
  • Platelet Function Analyzer (PFA-100)

Buccal Mucosal Bleeding Time

• This is a test for primary hemostatic defects known as thrombopathia.
• Rule out thrombocytopenia via other clotting defects first.
• Using a specific lancet to make a standardized cut in the mucosa.
• Blood is absorbed with filter paper, and the results of this may be affected by sedation and anesthesia.
• BMBT is the duration of time taken for a crescent of blood to stop forming on filter paper.
• This subjectivity gives it poor reproducibility/insensitivity.
• Anemia may prolong the effect.
• Differentials for prolonged BMBT consist of thrombocytopenia, thrombopathia, and Von Willebrand's disease.

Dog Breed Variations

• Lower platelet counts can occur in some dog breeds:
  • Greyhound (80,000-100,000/μL)
  • Shiba Inu and Akitas have macrothrombocytopenia.
  • Cavalier King Charles Spaniels and others (Norfolk and Cairn terriers) also have inherited macrothrombocytopenia.

Inherited Macrothrombocytopenia

• Cavalier King Charles Spaniels, Norfolk, and Cairn Terriers may be affected.
• Characteristics are large platelets (↑ MPV), mild to marked thrombocytopenia, and no increased bleeding risk.
• This is due to a mutation in β1-tubulin, and a definitive diagnosis comes from a genetic test.
• Genetic testing is helpful if unsure whether thrombocytopenia is inherited or due to illness.

Quantitative Platelet Disorders

• Decreased platelets: Thrombocytopenia
• Increased platelets: Thrombocytosis

Influences on Platelet Count

• Influences consist of:
  • Production
  • Consumption
  • Destruction
  • Sequestration or release from spleen & lungs

Causes of Thrombocytopenia

• Causes of thrombocytopenia include:
  • Pseudothrombocytopenia, which needs to be ruled out with blood film evaluation.
  • Decreased production
  • Increased consumption
  • Increased destruction
  • Blood loss (less common)
  • Sequestration specifically in the spleen (splenomegaly) and lungs (mild thrombocytopenia)

Thrombocytopenia-Induced Bleeding

• Spontaneous hemorrhage due to thrombocytopenia does not occur unless the platelet count is markedly decreased.
  • < ~30,000/μL
  • 30,000-50,000 increases the bleeding risk
• Hemorrhage does not cause marked thrombocytopenia

Pseudothrombocytopenia

• This is a false decrease in platelet count, most commonly caused by platelet clumping.
• Difficult venipuncture can result in platelet activation, especially in cat, rabbit, and cattle platelets, as well as EDTA-induced clumping.
• Detection of it is via:
  • Evaluation of the blood film for platelet clumps
  • Check sample tube for clumps/clots

Thrombocytopenia: Decreased Production

• Patients will frequently demonstrate additional cytopenias
• This can be caused by damage to stem cells or microenvironment.
• Also can be caused by suppression of thrombopoiesis or hematopoiesis, and these can cause moderately to markedly decreased platelet counts.

Causes of Decreased Production

• Can be caused by:
  • Drug-induced/toxic substances:
    • Cytotoxic chemotherapy
    • Idiosyncratic (sulfa drugs, methimazole, gold salts, penicillin)
    • Estrogen (therapy, Sertoli cell tumors)
    • Bracken fern in ruminants
  • Infectious diseases:
    • Bovine viral diarrhea virus
    • Distemper
    • Ehrlichiosis, other rickettsial
    • Equine infectious anemia (EIA)
    • FeLV
  • Bone marrow neoplasia or fibrosis (myelophthisis)
  • Aplastic anemia
• A thorough drug history is recommended.

Thrombocytopenia: Consumption

• Increased utilization or activation of platelets can also cause it, and causes mild to moderate thrombocytopenia (~60,000-100,000/µl).
• Causes consist of:
  • Vascular lesions such as:
    • Internal trauma
    • Vasculitis: A. phagocytophilum, RMSF, FIP, SLE, etc.
    • Vascular neoplasia: Hemangiosarcoma
  • Increased activation of coagulation such as:
    • Disseminated intravascular coagulation (DIC)
    • Snake envenomation (rattlesnakes)
    • Inflammation
    • Heat stroke
• Similar to Virchow's triad of thrombosis.

Thrombocytopenia: Destruction

• Immune-mediated disease also known as (ITP/IMTP/IMT) can be primary or secondary.
• Generally associated with marked thrombocytopenia with <30,000/μL.
• Dogs are more affected than cats or horses.
• Primary destruction is from anti-platelet antibodies (autoimmune or idiopathic).
• Secondary destruction is from antibodies directed against non-self antigens present on the surface of platelets.
• This can come from drugs or vaccines or infectious agents like Anaplasma platys or paraneoplastic syndrome like lymphoma.

ITP: Diagnosis

• Patients typically have very low platelet counts, often <10,000/μL.
• Diagnosis is most commonly done by excluding other diseases and/or by the response to therapy.
• Antiplatelet antibody testing is currently not readily available/expensive.
• The hallmark of diagnosis is marked thrombocytopenia in the patient.

Hemorrhage-Induced Thrombocytopenia

• Hemorrhage does not always cause thrombocytopenia.
• When it does, is from acute or severe hemorrhage.
• Generally, it's usually mild to moderate
• Associated with platelet loss and consumption.
• Platelets are released from storage pools in spleen and lung.

Causes of Thrombocytosis

• Physiologic
• Reactive
• Neoplastic

Physiologic Thrombocytosis

• This is mild and transient.
• May be associated with "physiologic leukocytosis."
• It's caused by splenic contraction, where a normal spleen contains about 1/3 of platelets.
• Epinephrine, exercise, and pregnancy can cause it.

Reactive Thrombocytosis

• Is promoted by thrombopoietin due to inflammation.
• Can also be related to neoplasia like lymphoma, or from mast cell tumors, or from iron deficiency/hemorrhage (possibly due to erythropoietin crosstalk), or from endocrinopathies like hyperadrenocorticism, diabetes mellitus, and hypothyroidism.
• "Rebound" after thrombocytopenia, it can also be triggered by drug-related issues like vinca alkaloids/vincristine, corticosteroids, beta-adrenergic drugs i.e. epinephrine.
• Can occur after Post-splenectomy

Neoplasia of Platelets/Thrombocytes

• Essential thrombocythemia is a rare condition.
• Characterized by persistent and marked neoplastic thrombocytosis.
• Patients may have both abnormal morphology and function.
• Platelet counts are typically over 1 million/µL, even though those with PLT counts >1 million/µL can be seen with reactive causes.
• Acute megakaryoblastic leukemia (AML-M7) is rare.
• Characterized by circulating nucleated neoplastic cells (megakaryoblasts).
• Anemia +/- thrombocytopenia and metarubricytosis is possible.
• Tends to impair bone marrow function.

Platelet Function Defects

• AKA is also named thrombopathia, and displays abnormal primary hemostasis with adequate platelet numbers.
• It shows the same clinical signs as thrombocytopenia such as petechiae and mucosal bleeding.
• Secondary hemostasis isn't affected.

Von Willebrand Disease

• The most common hereditary platelet disorder in dogs.
• It especially affects the Doberman Pinscher, German Shepherd Dog, German Shorthaired Pointer, Golden Retriever, Scottish Terrier, Shetland Sheepdog and Standard Poodle.
• Also described in horses, cats, cows, pigs, and mice.

Von Willebrand Disease (vWD)

• Is deficiency in functional VWF (abnormal amounts +/- multimer distribution)
• Type 1 is low VWF concentration with normal multimer distribution, and is a common type.
• Type 2 is low VWF concentration with lower proportion of high-molecular-weight multimers which can be severe
• Type 3 is the almost total lack of VWF (most severe – mutation of VWF gene).
• It results in decrease platelet function.
• Consists of: Adhesion and aggregation functions.

Diagnosis of von Willebrand Disease

• This is done via the von Willebrand antigen test (VWF Ag).
• Consists of a ELISA or latex immunoassay where Results are reported as "% normal."
• <50% indicates VWF deficiency.
• The Multimeric analysis (immunoelectrophoresis) looks at the relative amounts of different sized multimers and is necessary to distinguish Type 1 vs. Type 2.
• With functional VWF assays, type 2 VWD could be detected.

Other Inherited Platelet Dysfunctions

• Adhesion receptor defects:
  • GPIIb-IIIa defect or deficiency (Glanzmann thrombasthenia) in Great Pyrenees, Otterhound, and those in horses.
• Signal transduction defects
  • Results in lack of activation of GPIIb/IIIa in Spitz, Basset Hound, Simmental cattle, cats and fawn-hooded rats.
• Storage granule defects:
  • Chediak-Higashi (Persian cats, cattle, beige rats) and dense granule pool disease of Cocker Spaniels (decreased ADP in platelets).

Acquired Platelet Dysfunction

• From Drug-induced causes :
  • NSAIDs/Aspirin- inhibits platelet cyclooxygenase to block thromboxane A2 synthesis
  • Antibiotics and penicillins surface binding
  • Antiplatelet drugs, such as clopidogrel
• Non-drug-induced causes
  • Uremia
  • CKD
  • Dysproteinemias
  • Snake envenomation
  • Neoplasia
• Proteins from cancer coats platelets and prevent them working properly