Platelets

Hemostasis

• interactions of proteins, cells, & blood vessels to stop bleeding when there is injury

• maintains blood in fluid state in vessel & allows for rapid formation of clot when injured

• need balance to avoid excess bleeding/clotting

  • deficient coagulation = hemorrhage

  • excess coagulation = thrombosis

Main contributors to hemostasis

• vessel wall/endothelium

• platelets

• coagulation cascade/factors

Steps of hemostasis

• vasoconstriction

• primary hemostasis - blood vessel wall & platelets form unstable plug

• secondary hemostasis - coagulation factors stabilize plug

• thrombus formation

• fibrinolysis - breaks down clot, contains hemostasis

Steps of primary hemostasis

• platelet adhesion to damaged subendothelial matrix

• platelet activation (change shape, release granules, attract/recruit more platelets)

• platelet plug formation

  • unstable, held together by soluble fibrinogen will dislodge if not stabilized

Thrombosis

• pathological state of hemostasis resulting in formation of inappropriate clots in vessles

• injured/activated endothelium can change from anticoagulant to procoagulant

Virchow’s triad

• endothelial injury

• abnormal blood flow (stasis or turbulence)

• hypercoaguability (inherited or acquired)

all contribute to thrombosis

Normal endothelial cells

• help maintain balance of hemostasis

• barrier between platelets & subendothelial matrix to prevent over-clotting

• produce Von Willebrand factor, tissue factor, & plasminogen activator inhibitors during vascular damage

Anticoagulant properties of endothelium

• primary: inhibit platelet adhesion/aggregation (platelets can’t attach to materials under endothelium), secretes prostacyclin & nitric oxide for vasodilation

Procoagulant properties of endothelium

• primary: produces Von Willebrand factor - cofactor for platelet binding after endothelial injury

Normal platelet morphology

• clear membrane bound fragments of cytoplasm, anucleic

• size varies with species, normally smaller than RBCs

• fine red-purple granules

• predisposed to clumping together towards feathered edge (especially cats)

Normal platelet function

• cells involved in mammalian hemostasis

• form a clot/plug in vessels to stop hemorrhaging

Thrombocytes

• equivalent to platelets in birds/reptiles

• oval to round, smaller than RBC, clear-faint blue cytoplasm, round nucleus w/clumped chromatin

• 1-3 per 100x field normal

Platelet granules contents

• molecules that promote activation of other platelets (agonists)

• molecules, coagulation factors, & co-factors used to form a clot

Adhesion molecules

• glycoprotein 1b - binds vWf, bridges platelets to subendothelial collagen

• glycoprotein 2b/3a - binds platelets to fibrinogen to form plug

Platelet production

• from megakaryocytes in bone marrow, may be sequestered/stored in spleen

  • EMH from spleen, lymph nodes, liver

• circulate 5-7 days, consumed in hemostasis or phagocytized by macrophages

Laboratory evaluation of primary hemostasis

• plate Ct-instrument: automated platelet count

• MPV: average size of platelets

• platelet eval: estimate of number on slide

• clumps (y/n)

Collection tubes

• with anticoagulant

• purple top w/EDTA or blue top w/sodium citrate

Reduce platelet clumping

• rapid blood draw, reduce patient struggling

• place blood in anticoagulant tube ASAP (detach needle)

Quantitative platelet evaluation

• automated analyzers most accurate

• manual count w/hemocytometer less accurate, not same as platelet estimation

• estimate: blood smear monolayer examined at 100x, average over multiple fields multiplied by 20 × 10^9/L

• clumps can artificially decrease platelet counts

Low platelet risks

• moderately decreased: risk of hemorrhage after biopsy of vascular organ

• markedly decreased: risk of spontaneous hemorrhage

Idiopathic congenital macrothrombocytopenia

• inherited, common in Cavies & Norfolk/Cairn terriers

• mutation causes defective fragmentation of megakaryocyte cytoplasm, produce fewer & larger platelets that still have normal function

  • low platelet count, high MPV, normal PCT

Disorders of primary hemostasis

• most common cause: low platelet numbers

  • other reasons: impaired platelet function, vascular disease

• clinical signs: petechia (small red spots), ecchymoses (1+ cm red spots/bruises), spontaneous mucosal bleeding

Evaluation for disorders of primary hemostasis

• determine total platelet count

• BMBT test if count normal

Evaluation of platelet function

• can be abnormal even if numbers are normal, leading to disorder of primary hemostasis

Buccal mucosal bleeding time

• bedside test for primary hemostasis

• spring loaded cassette delivers cut of precise depth/length

• evaluate time until bleeding stops

  • 1-5 minutes (dogs), 1-3.5 minutes (cats)

• only perform test if platelet number is known but function is in question

  • should be over 100 × 10^9/L

Thrombocytopenia

• most common cause of primary hemostatic disorder

• causes:

  • consumption - utilized during hemostasis

  • destruction - immune-mediated phagocytosis

  • decreased production - bone marrow damage

Consumption thrombocytopenia

• hemostasis secondary to hemorrhage (regenerative anemia)

• disseminated intravascular coagulation/excessive clot formation (abnormal coags)

• vasculitis/blood vessel inflammation exposing subendothelial matrix (± inflammatory leukogram)

Destruction thrombocytopenia (ITP)

• most common cause: immune-mediated

  • immune system produces antibodies against own platelets/megakaryocytes, phagocytized by macrophages in liver/spleen

  • most common in female dogs, breed disposition

• may be idiopathic or secondary to vaccines/drugs/infections

• marked thrombocytopenia, increased MPV, increased megakaryocytes in marrow (if not targeted as well)

  • ± Evans Syndrome (concurrent IMHA)

• diagnosis of exclusion

Decreased production thrombocytopenia

• causes:

  • myelopthisis - replacement of hematopoietic cells in marrow

  • infection of megakaryocytes - FIV, FeLV, distemper, EIA, Erlichia, BVDV

  • drugs/toxins

  • irradiation

• moderate to marked thrombocytopenia, no macroplatelets, decreased megakaryocytes in bone marrow, often bi/pancytopenia

Von Willebrand disease

• most common inherited bleeding disorder

• defective and/or deficient vWF, causes defect of platelet adhesion

  • defect of primary hemostasis

• signs: prolonged/mucosal bleeding, usually with normal platelet count + coag tests

Rare inherited defects of platelet function

• Chediak-Higashi - abnormal granulation, do not aggregate

• Glanzmann thrombasthenia - abnormal/deficient Gp2b-3a receptor, do not aggregate

• Bernard-Soulier - abnormal/deficient Gp1b, platelets do not bind vWF to adhere to subendothelium

Acquired platelet function defects

• drugs (NSAID/aspirin) inactivate platelet COX & block TXA2 production

• TXA2 needed to activate Gp2b/3a (fibrinogen binding site), platelets will not aggregate

Thrombocytosis

• increased platelets

• common: may be physiologic (epinephrine) or reactive (production from inflammation)

• rare: neoplastic proliferation

Physiologic thrombocytosis

• release of epinephrine causes splenic contraction

• temporary movement of splenic platelets into circulation - transient increase

• platelet production/megakaryocytes not increased

Reactive thrombocytosis

• increased production of platelets, increased megakaryocytes in marrow

  • often increased MPV

• mediated by thrombopoietin in health, inflammatory cytokines stimulate production during inflammation

• often concurrent with iron deficiency anemia

Essential thrombocythemia

• rare neoplastic proliferation of platelets

• marked increase of platelets (1 million/uL +)

• increased megakaryocytes in bone marrow

Secondary hemostasis steps

• injured endothelium releases tissue factor/factor III

• shape change (2D → 3D) via membrane phospholipids coming to surface

• activated coagulation factors (thrombin/II) bind to platelet surface

• conversion of fibrinogen to insoluble cross-linked fibrin that holds platelets from primary plug together

Coagulation factors

• proteins made in liver, circulate in inactive form

• intrinsic & extrinsic pathways converge at common pathway

Vitamin K dependent factors

• II, VII, IX, X

• carboxylation does not occur without, can’t bind Ca2+ or platelet surfaces

• deficiency = severe hemorrhagic disorder

Extrinsic vs intrinsic pathway

• initiates coagulation, forms small amount of thrombin (II)

• tissue factor (III) activates CF VII, both activate CF X (common pathway)

vs

• amplifies coagulation, forms large amount of thrombin (II)

• XII, XI, IX, VIII activate CF X (common)

Common pathway

• CF X & V (cofactor) convert prothrombin to thrombin (II)

• thrombin converts fibrinogen to fibrin & activates CF XIII

• CF XIII cross-links fibrin to non-soluble fibrin polymer (very stable, hard to degrade)

  • technically not part of pathway

Preventing uncontrolled clotting

• restrict hemostasis to platelet surfaces

• blood flow prevents accumulation of activated CF

• activated CF removed by macrophages in liver or destroyed by endothelial coagulation inhibitors

• fibrinolytic system dissolves clot

Antithrombin III

• protein made in liver, circulates as inactive

• when endothelium injured, heparin expressed & activated

• binds to & inhibits thrombin + CF, IX, X, XI

• decrease = hypercoaguable

  • causes: decreased production (liver disease), increased consumption (DIC), protein loss

Tissue factor pathway inhibitor

• inactivates TF:VII complex & CF X

Thrombomodulin

• inactivates thrombin

Proteins C & S

• inactivate factors V & VIII

• vitamin K dependent

Fibrinolysis

• enzymatic process by which fibrin clot is lysed

• concomitant with coagulation

• prevents unchecked clotting

Plasmin

• fibrinolytic enzyme (fibrinogen, soluble + insoluble fibrin)

• inactivates CF XIII

• circulates in inactive form, activated by tissue plasminogen activator

Fibrin(ogen) degredation products (FDPs)

• from breakdown of fibrinogen + soluble fibrin by plasmin

• measurable in blood, cleared by liver in health

• agglutination test to evaluate

D-Dimers

• from breakdown of cross-linked fibrin

Increased FDP & D-Dimers

• secondary to excessive fibrinolysis (DIC, hemorrhage, thrombosis)

• decreased clearance from liver (liver disease, macrophage abnormalities)

Secondary hemostasis blood tests

• activated clotting time - intrinsic/common

• partial thromboplastin time - time to fibrin formation, intrinsic/common

  • needs <30% to prolong

  • no effect from thrombocytopenia

• prothrombin time - time to fibrin, extrinsic/common

  • needs <30% to prolong

  • no effect from thrombocytopenia, sensitive to vitamin K antagonism (short half-life of CF VII)

• thrombin time - fibrinogen → fibrin

• use blue top/sodium citrate tube (less effective at binding Ca2+)

Coagulation panel

• PT + PTT

• other tests special ordered, ACT cage-side

Disorders of secondary hemostasis

• common signs: hematoma, bleeding into body cavities, delayed hemorrhage

Hemophilia

• inherited secondary hemostatic defect

• deficiency of clotting factor causing mild to severe hemorrhage

  • A = CF VIII

  • B = CF IX

  • C = XI

• show as prolonged aPTT

Inherited defects of extrinsic pathway

• deficiency/defect of CF VII

• show as prolonged PT

Inherited defects of common pathway

• disorders of CF X, thrombin, fibrinogen

• disorder of CF II incompatible with life

• shows as increased PT + aPTT, may have increased BMBT with fibrinogen deficiency

Acquired defects of secondary hemostasis

• more common

• vitamin K deficiency or liver disease

Vitamin K deficiency

• causes: decreased production, malabsorption, dietary deficiency, antagonism (ingestion of anticoagulants)

  • inhibit epoxide reductase

• signs: life-threatening hemorrhage, epistaxis, hemoptysis, lethargy, respiratory distress, lameness

• PT, aPTT, & ACT prolonged, PT increased first

Liver disease

• failure to produce coagulation factors (including fibrinogen) & antithrombin III

• PT, aPTT, ACT, TT, BMBT all prolonged

Disseminated intravascular coagulation

• mixed hemostatic disorder, defects in primary + secondary hemostasis & fibrinolysis

• increased activation + consumption of CF, antithrombin III, & platelets, thrombosis → hemorrhage

• highly deadly, always secondary to another pathology (ex: sepsis)

• prolonged PT, aPTT, ACT + thrombocytopenia