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Hemostasis/Coagulation. Gregory S. Travlos, DVM, DACVP National Institute of Environmental Health Sciences Research Triangle Park, NC 27709 919-541-0653 Travlos@niehs.nih.gov. Hemostasis. The process by which bleeding is arrested.

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Hemostasis/Coagulation

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Hemostasis/Coagulation

Gregory S. Travlos, DVM, DACVP

National Institute of Environmental Health Sciences

Research Triangle Park, NC 27709

919-541-0653

Travlos@niehs.nih.gov


Hemostasis

The process by which bleeding is arrested.

  • It is a series of physiological and biochemical events which terminate in the formation of an insoluble fibrin clot

    Hemostatic Sequence:

  • Interaction between vessel wall and platelets

  • Blood coagulation

  • Fibrinolysis


Hemostatic Component Interactions

Thompson &

Harker, 1983


Blood Vessels

Intact endothelium forms a thromboresistant surface

  • Required for the free flow of blood; does not promote platelet adherence or activate coagulation

  • Passive mechanisms:

    • Endothelial glycocalyx (negative charge - repels like-charged particles, e.g.,platelets).

    • Presence of a2-macroglobulin at cell surface (protease inhibitor).

  • Active mechanisms:

    • Endothelial cells remove platelet aggregation promoters from circulation (e.g., PGF1, bradykinin, serotonin, adenine nucleotides).

    • Secretion of PGI2 - potent inhibitor of platelet aggregation, induces vasodilation.

      Proteoglycan matrix of the vessel wall influences thrombogenicity.

  • Heparin, heparan sulfate and dermatan sulfate have anticoagulant activity; other glycosaminoglycans and hyaluronic acid do not.

  • Veins have the highest concentration.


Endothelium

Besides their role in thromboresistance, endothelial cells have additional synthetic functions.

  • Produce Von Willebrand’s factor

    • Absorbed by platelets; needed for adherence to collagen

  • Produce plasminogen activator (tPA)

    • Mediates fibinolysis

  • Injured cells release thromboplastin (factor III)

    • Activates the “extrinsic” coagulation cascade

  • Others (e.g., type III and IV collagens, elastin, fibronectin, etc.)


Blood Vessel Structure

Thompson &

Harker, 1983


Platelets

Adhere to exposed collagen (platelet plug)

  • Occurs in seconds; can control hemorrhage of minute injuries

    Secretory functions; mediators of coagulation and fibrinolysis

  • Releases ADP; sticky and promotes platelet adherence

  • ADP activates phospholipase A2 which stimulates thromboxane A2 synthesis

  • Release of membrane fibrinogen, factor V, factor VIII and calcium

  • Release of membrane platelet phospholipid.


microtublules

OCS

granules

mitochodrion

Platelet - TEM


Ultrastructural and Functional Platelet Anatomy


Platelets - cont.

The role of platelets in hemostasis is as important as the coagulation mechanism.

  • Thrombocytopenia, thrombasthenia or thromobopathia - impair hemostasis

  • Thrombocytosis or thrombocythemia - may impair, but usually promotes clotting (predisposes to thrombosis).

    Platelets promote hemostasis by:

  • Release of ADP and other agonists; promotes adherence.

  • ADP activates phospholipase A2 which stimulates thromboxane A2 synthesis

    • Thromboxane A2 - stimulates vasoconstriction and platelet aggregation

  • Release of membrane fibrinogen, factor V, factor VIII and calcium

    • Components of coagulation localized at site of injury

  • Release of membrane platelet phospholipid.

    • Accelerates the “intrinsic” and “common”pathways of coagulation


Prostaglandin Metabolism

Harlan &

Harker, 1981


Hemostatic Platelet Functions

Thompson &

Harker, 1983


Platelet Response

When a vessel is injured or severed a brief, local, reflex vasoconstriction occurs.

  • Reduces blood flow at site.

  • Maintained by vasoactive compounds (platelets, surrounding tissues).

    Passing platelets adhere to exposed collagen.

  • Occurs in seconds; initially adhere in a single layer and become activated.

  • Severe injury - collagen serves as a potent platelet activator.

  • Less severe injury - vWF and fibrinogen become the major activators.

    The adhered platelets undergo a conformational change.

  • From discoid to development of long filopodia.

  • Activation of GP receptors for fibrinogen and/or vWF (GPIIb/IIIa and GPIb/IX/V).


Structure of the GPIb-IX-V receptor

Tablin, 2000


Platelet Response to Agonists

Platelets - unstimulated

Addition of ADP

(mild stimulation)

Addition of thrombin

(strong stimulation )

Characteristic discoid shape

Shape change (elongation and crescents) and filaform process formation (arrows)

Increased spreading, filaform process extension (arrows) and aggregate formation (stars)

SEM plates;

Gentry, 2000


Platelet Response cont.

Activated platelets release their a-granule and dense body contents inducing additional platelet recruitment.

  • Dense granules - ADP, serotonin and epinephrine.

  • alpha-granules - fibrinogen (and vWF in human and pig).

  • Synthesis and release of PAF and TxA2.

    The agonists accelerate the development of an irreversible platelet aggregate (platelet plug).

  • Reversible v. irreversible responses.

  • Thrombocytes of birds and reptiles do not respond to ADP.

  • Serotonin and epinephrine:

    • Serotonin - shape change (rat, g. pig and dog); aggregation (human, rabbit, cow, horse, pig, sheep and cat).

    • Epinephrine - only human, primate, cat and horse platelets appear responsive.

    • Either serotonin or epinephrine combined with another agonist - strong response in all species.


Platelet Response cont.

More about agonists.

  • Platelet Activating Factor (PAF).

    • Cow, horse, sheep, primate, dog, g. pig and rabbit respond to PAF.

    • Human less sensitive and rat and mouse are insensitive to this agonist.

  • Thromboxane A2 (TxA2).

    • Strong agonist - human,g. pig and rabbit.

    • Weak agonist - horse.

    • Insensitive - rat, cow, pig.

      In real life, however, platelets are exposed to multiple agonists from platelets and other cells (e.g., red cells, ADP; white cells, PAF).


Platelet Aggregation to Thrombin

Harlan &

Harker, 1981


Hemostatic Plug Formation

Baumgartner

& Muggli, 1980


Coagulation System

Consists of a cascading system of proteins

  • Primarily originating from liver (except factor III)

  • Circulate in inactive form (except, possibly, factor VII)

  • System includes:

    • Enzymatic factors

    • Non-enzymatic factors

    • Tissue thromboplastin (factor III)

    • Calcium (factor IV)

    • Platelet phospholipid (PF 3) - structural component; accelerates factor activation

    • Anticoagulant factors

      The coagulation system consists of three pathways (intrinsic, extrinsic and common)


Procoagulant Factors


Coagulation Systems - cont.

Enzymatic factors

  • Circulate as non-active zymogens - must be activated to function

  • Activated enzymatic factors are not consumed during clotting (except factors II and XIII)

  • Partial deficiency results in partial loss of clotting ability

  • Activated enzymatic factors inhibited by antithrombin III (complexed with heparin) and some alpha-2-glycoproteins

  • Enzymatic factors:

    • XI and XII (contact factors)

    • II, VII, IX and X (vitamin K-dependent factors)

    • XIII (clot stabilizing factor or fibrin-stabilizing factor)


Coagulation Systems - cont.

Non-enzymatic factors

  • Originate from liver but associate with platelet membranes (also found in plasma)

  • Normal clotting with partial deficiency; almost total absence needed to affect hemostasis or clotting

  • Clotting consumes these factors - absent in serum

  • No known natural inhibitors

  • Considered reactive proteins - increased during inflammatory and neoplastic processes (except factor III)

  • Non-enzymatic factors:

    • Fibrinogen (factor I)

    • Factor V

    • Factor VIII:C (associated with Von Willebrand’s factor)


Coagulation Cascade Interactions


Of course, he does

But, his feathered companion does not

Does this turkey have factor XII?


Coagulation Systems - cont.

Clot stabilization

  • Fibrin stabilizing factor (factor XIII) forms fibrin strand cross-links.

  • Synthesized by monocytes and hepatocytes.

  • Zymogen is activated by thrombin (plus calcium).

  • A very small amount of factor XIII (2 - 10%) is adequate for hemostasis.

  • Converts soluble fibrin monomers (unstable) to a fibrin polymer (stable).

  • Lead, silver, zinc and snake venoms are known inhibitors.


Coagulation Inhibitors

The activity of coagulation system must be attenuated.

  • Numerous inhibitors are found in blood.

    Coagulation is controlled by three types of actions.

  • Inhibition of converting enzymes (e.g., AT III, C1 esterase inhibitor, a2-macroglobulin, a2-antiplasmin, a1-antitrypsin, HC-II).

    • Act on one or more of the converting enzymes (activated factors).

    • AT III-heparin pathway: major system - 80% of the thrombin inhibitory action in plasma.

  • Destruction of protein cofactors (e.g., TM-PC-PS system).

    • TM-PC-PS system degrades cofactors V & VIII:C, inhibiting prothrombinase and tenase complexes, respectively.

  • Blocking receptor availability needed for complex formation (e.g., Tissue factor pathway inhibitor (TFPI) and annexin V).


AT III

Th

H

AT III

Proposed Mechanism of AT III-Heparin System

Lysine sites

H

Serine site

Arginine

site

Heparin

Thrombin

Antithrombin III

Th


Proposed Mechanism of Thrombomodulin, Protein C and Protein S (TM-PC-PS) System

F-Xa

Prothrombin

Activated

platelet

F-Va

PS

Thrombin

x

Ca++

Ca++

Protein C

Activated

Protein C

Thrombin

Thrombomodulin


Proposed Mechanism of Tissue Factor Pathway Inhibitor (TFPI) Activity

F-Xa

F-Xa

TFPI

TFPI

F-Xa

TFPI

Tissue factor

F-VIIa

Endothelium


Anticoagulant Factors


Fibrinolytic System

Method for removing clots and maintenance of a patent vascular system and fibrin deposited during inflammation and tissue injury must be removed.

  • Plasmin (serine protease) primarily responsible for fibrinolysis.

  • Produced in the liver and kidney, it circulates in an inactive form (plasminogen).

    • Activators: tissue plasminogen activator (tPA), cytokinases-urokinases (urine, CSF, tears, saliva, milk, bile, synovial, prostatic and amniotic fluids), erythrocyte erythrokinase, neutropil activator and factor XII-dependent activator (XII-prekallikrien-hageman factor cofactor complex).

  • In addition to fibrin and fibrinogen, plasmin will hydrolyse a variety of proteins.

  • While plasminogen is normally found in blood and body fluids, plasmin is usually absent due to numerous antiplasmins.

    • Inactivators: antithrombin III, a2-macroglobulin, a1-antitrypsin and C1 inactivator.


Activation

Inhibition

Fibrinolytic System and Factors Regulating Fibrinolysis (Fibrinogenolysis)

Plasminogen

Damaged

endothelium

Kallikrein

Plasminogen activator inhibitor

e-aminocaproic acid

FHIIa

tPA

Prekallikrein

Streptokinase

Urokinase

Plasmin

a2-Antiplasmin

a2-Macroglobulin

Biodegradation of

FV, FVIII, FIX, FXI

fibrinogen

Complement activation

Fibrin/fibrinogen

Degradation products

Firbrinogen/fibrin


Fibrinogen or Fibrin

Fragment X

Small Peptides

Fragment Y

Fragment D

Small Peptides

Fragment E

Fragment D

Small Peptides

Degradation of Fibrin/Fibrinogen

Plasmin

Plasmin

Plasmin


Evaluation of Hemostasis

Fundamental physiology and pathophysiology of hemostasis is similar in mammalian species.

  • Variables identical for laboratory animals and human patients

    Platelets

  • Platelet count - detection of thrombocytopenia

  • Clot retraction - non-anticoagulated blood

    • Failure to separate - platelet function defect or thrombocytopenia

  • Bleeding time (BT)- in vivo test; simple; low sensitivity

    • Used to evaluate platelet function defects

    • Thrombocytopenia - prolongs BT

    • Clotting factor deficiency does not alter BT

    • Vascular disease (eg., scurvy) can prolong BT (humans, guinea pigs)


Considerations for Blood Collection

Clean/smooth surfaces

  • Want to avoid platelet clumping or activation of factor XII

    • Use plastic or siliconized glass for sample collection

    • Animal blood clots faster than human blood - prime needle with anticoagulant

      Collect sample from an endothelial-lined vessel and careful venipuncture

  • Want avoid contamination with tissue juice (factor III)

  • Small clot activates coagulation system invalidating results

  • Samples from indwelling catheters are usually unacceptable


Sample Handling/Anticoagulants

Plasma samples separated from cells within 30 minutes

  • Perform analyses immediately

  • Plasma samples may be quickly frozen (dry ice/alcohol or liquid nitrogen) and stored at -70o for analysis at a later date

  • Activity of factors V and VIII is lost rapidly in samples held at room temperature

    Citrate (trisodium salt) is the anticoagulant of choice.

  • Oxalate anticoagulants are acceptable - not commonly used

  • Heparin - unacceptable

  • EDTA - unacceptable (except for indirect evaluation of fibrinogen concentration by heat precipitation and refractometry)


Evaluation -cont.

Activated Coagulation Time (ACT) - in vivo test

  • Measures (seconds) time to clot formation in fresh whole blood

  • Careful attention to sample collection/handling

  • Platelet counts <10,000 cause slight increase in ACT

    • Results from lack of platelet phospholipid for test

  • Increased ACT suggests factor deficiency in intrinsic or common pathways

    • Deficiency must be 5% of normal to prolong ACT

      Activated Partial Thromboplastin Time (APTT)

  • Measures (seconds) time to clot formation in citrated plasma

  • Increased APTT - factor deficiency in intrinsic or common pathways

    • Deficiency must be 30% of normal to prolong APTT

    • Fibrinogen <50 mg/dL will prolong APTT; inflammation may shorten APTT

    • Sensitivity increased with saline-diluted plasma

    • Heparin therapy prolongs APTT - differentiate using a 1:1 dilution with normal plasma


Evaluation -cont.

One-Stage Prothrombin Time (OSPT, PT)

  • Measures (seconds) time to clot formation in citrated plasma

  • Rabbit or synthetic tissue thromboplastin preferred; human origin reagent gives longer PT times

  • Increased PT - factor deficiency in factor VII or common pathway

    • Deficiency must be 30% of normal to prolong PT

    • Fibrinogen <50 mg/dL will prolong PT

    • Sensitivity increased with saline-diluted plasma

      Russel’s Viper Venom Time (RVVT)

  • Measures (seconds) time to clot formation in citrated plasma

  • Increased RVVT - in or common pathway but insensitive to factor VII deficiency

    • Deficiency must be 30% of normal to prolong RVVT

    • Fibrinogen <50 mg/dL will prolong RVVT

    • Sensitivity increased with saline-diluted plasma


Evaluation -cont.

Thrombin Clotting Time (TCT)

  • Measures (seconds) time to clot formation in citrated plasma

  • Increased TCT - decreased fibrinogen concentration (<100 mg/dL), dysfibrinogenemia, increased FDP concentration, heparin therapy

    Fibrinogen Concentration (factor I)

  • In most species, fibrinogen is 100 - 400 mg/dL

  • Fibrinogen decreases in DIC, severe liver insufficiency and hereditary hypofibrinogenemia

  • Inflammation can increase fibrinogen concentration


Evaluation -cont.

Fibrin-Fibrinogen Degradation Products (FDP)

  • Measures, by latex agglutination, the concentration of products of fibrinolysis; D-dimer assay is another method for measuring FDP

  • Increased FDP - occurs with disseminated intravascular coagulation or severe internal bleeding

  • In most species, normal FDP is <10 micrograms/mL


Example

Acute oral study in dogs

Animals given 3 X LD50 in food

  • Brodifacoum

  • Bromadiolone

  • Diphacinone

    Coagulation studies

  • ACT

  • RVVT

  • PT


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