Hemostasis/Coagulation

Gregory S. Travlos, DVM, DACVPNational Institute of Environmental Health Sciences

Research Triangle Park, NC 27709919-541-0653

Travlos@niehs.nih.gov

HemostasisThe 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 VesselsIntact 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 2-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.

EndotheliumBesides 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

PlateletsAdhere 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.

Platelet - TEM

mitochodrion

microtublules

OCS

granules

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

Platelet ResponseWhen 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).

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 -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 SystemConsists 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

Does this turkey have factor XII?

Of course, he does

But, his feathered companion does not

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 InhibitorsThe 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, 2-

macroglobulin, 2-antiplasmin, 1-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).

Coagulation InhibitorsCoagulation is controlled by three types of actions.

• Inhibition of converting enzymes (e.g., AT III, C1 esterase inhibitor, 2-macroglobulin, 2-antiplasmin, 1-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).

Proposed Mechanism of AT III-Heparin System

HeparinThrombin Antithrombin III

Lysine sites

Serine site

Argininesite

H

Th

H

AT III

AT III

Th

Coagulation InhibitorsCoagulation is controlled by three types of actions.

• Inhibition of converting enzymes (e.g., AT III, C1 esterase inhibitor, 2-macroglobulin, 2-antiplasmin, 1-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).

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

Thrombin

Prothrombin

Protein C

Thrombomodulin

Thrombin

F-Xa

Activatedplatelet

PS

F-Va

Ca++

Ca++

ActivatedProtein C

Activated PC associates with PS and degrades cofactors V and VIII:C

Coagulation InhibitorsCoagulation is controlled by three types of actions.

• Inhibition of converting enzymes (e.g., AT III, C1 esterase inhibitor, 2-macroglobulin, 2-antiplasmin, 1-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).

Proposed Mechanism of Tissue Factor Pathway Inhibitor (TFPI) Activity

F-Xa

Endothelium

Tissue factorF-VIIa

TFPI

F-Xa

TFPITFPI

F-Xa

Anticoagulant Factors

Fibrinolytic SystemMethod 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, 2-macroglobulin, 1-antitrypsin and C1 inactivator.

Fibrinolytic System and Factors Regulating Fibrinolysis (Fibrinogenolysis)

PlasminogenActivation Inhibition

Damagedendothelium

Kallikrein

Plasminogen activator inhibitor

-aminocaproic acid

Prekallikrein

Streptokinase

Urokinase

FHIIa

tPA

Plasmin

2-Antiplasmin

2-Macroglobulin

Complement activation

Fibrin/fibrinogenDegradation products

Biodegradation of FV, FVIII, FIX, FXI fibrinogen

Firbrinogen/fibrin

Degradation of Fibrin/Fibrinogen

Fibrinogen or Fibrin

Fragment X Small Peptides

Fragment Y Fragment D Small Peptides

Fragment E Fragment D Small Peptides

Plasmin

Plasmin

Plasmin

Evaluation of HemostasisFundamental 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 CollectionClean/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/AnticoagulantsPlasma 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

Hemorrhagic Anemia Causes• Bleeding lesions

• Gastric ulcers (aspirin)

• Coagulation disorders• Anticoagulants

• Direct acting (heparin) • Altered clotting factor production (coumarins, indanediones)

• Disseminated intravascular coagulation (DIC)

• Thrombocytopenia• Immune-mediated (methyldopa, phenacetin)• Nonimmune-mediated (ristocetin)• Decreased production (chemotherapeutic drugs)• Increased consumption - DIC

Coumarin-type AnticoagulantsAcute (14-day) oral study in dogs

Animals given 3 X LD50 in food • Bromadiolone

CBC and Coagulation studies • Activated Clotting Time (ACT) - extrinsic pathway

• Could also use activated partial thromboplastin time (APTT)

• Prothrombin Time (PT) - intrinsic pathway• Russell’s Viper Venom Time (RVVT) - common pathway

Mechanism of action of vitamin K- carboxylation of glutamic acid residues

PrecoagulantClotting Factors

(II, VII, IX, X)

CO2

Vitamin Kepoxide

FunctionalClotting Factors

(II, VII, IX, X)

Vitamin Kreduced

Vitamin K epoxide reductaseX

(Coumarins inhibit this enzyme)In hepatocytes

COMMON SYSTEM

(ACT, APTT) (PT)

(RVVT)COMMON SYSTEM

(ACT, APTT) (PT)

(RVVT)COMMON SYSTEM

Observations and CBC data: 14-day acute oral study of Bromadiolone in dogs

Clinical observations:Depression, vomiting and diarrhea by 5 days post-dosing

Observations and CBC data: 14-day acute oral study of Bromadiolone in dogs

Clinical observations:Depression, vomiting and diarrhea by 5 days post-dosingBleeding at venipuncture site as early as 6 days post-dosing

Observations and CBC data: 14-day acute oral study of Bromadiolone in dogs

Clinical observations:Depression, vomiting and diarrhea by 5 days post-dosingBleeding at venipuncture site as early as 6 days post-dosingBleeding at various sites (i.e., urinary tract, nose, gums, subcutaneous tissue -

hematomas, G. I. tract - blood/melena) occurred as early as 8 days post-dosing

Observations and CBC data: 14-day acute oral study of Bromadiolone in dogs

Clinical observations:Depression, vomiting and diarrhea by 5 days post-dosingBleeding at venipuncture site as early as 6 days post-dosingBleeding at various sites (i.e., urinary tract, nose, gums, subcutaneous tissue -

hematomas, G. I. tract - blood/melena) occurred as early as 8 days post-dosing

Pale mucous membranes observed at 11 days post-dosing

Observations and CBC data: 14-day acute oral study of Bromadiolone in dogs

Clinical observations:Depression, vomiting and diarrhea by 5 days post-dosingBleeding at venipuncture site as early as 6 days post-dosingBleeding at various sites (i.e., urinary tract, nose, gums, subcutaneous tissue -

hematomas, G. I. tract - blood/melena) occurred as early as 8 days post-dosing

Pale mucous membranes observed at 11 days post-dosing

CBC (means) day 0 day 7 day 11Hct (%) 48.3 43.4 26

Hgb (g/dL) 16.3 14.0 8.3

RBC (106/µL) 7.6 6.5 3.6

MCV (fL) 64 67 72

MCHC (g/dL) 33.8 32.3 32.0

Observations and CBC data: 14-day acute oral study of Bromadiolone in dogs

Clinical observations:Depression, vomiting and diarrhea by 5 days post-dosingBleeding at venipuncture site as early as 6 days post-dosingBleeding at various sites (i.e., urinary tract, nose, gums, subcutaneous tissue -

hematomas, G. I. tract - blood/melena) occurred as early as 8 days post-dosing

Pale mucous membranes observed at 11 days post-dosing

CBC (means) day 0 day 7 day 11Hct (%) 48.3 43.4 26 (anemia)

Hgb (g/dL) 16.3 14.0 8.3

RBC (106/µL) 7.6 6.5 3.6

MCV (fL) 64 67 72

MCHC (g/dL) 33.8 32.3 32.0

Observations and CBC data: 14-day acute oral study of Bromadiolone in dogs

Clinical observations:Depression, vomiting and diarrhea by 5 days post-dosingBleeding at venipuncture site as early as 6 days post-dosingBleeding at various sites (i.e., urinary tract, nose, gums, subcutaneous tissue -

hematomas, G. I. tract - blood/melena) occurred as early as 8 days post-dosing

Pale mucous membranes observed at 11 days post-dosing

CBC (means) day 0 day 7 day 11Hct (%) 48.3 43.4 26 (anemia)

Hgb (g/dL) 16.3 14.0 8.3

RBC (106/µL) 7.6 6.5 3.6

MCV (fL) 64 67 72 (macrocytic)

MCHC (g/dL) 33.8 32.3 32.0

Observations and CBC data: 14-day acute oral study of Bromadiolone in dogs

Clinical observations:Depression, vomiting and diarrhea by 5 days post-dosingBleeding at venipuncture site as early as 6 days post-dosingBleeding at various sites (i.e., urinary tract, nose, gums, subcutaneous tissue -

hematomas, G. I. tract - blood/melena) occurred as early as 8 days post-dosing

Pale mucous membranes observed at 11 days post-dosing

CBC (means) day 0 day 7 day 11Hct (%) 48.3 43.4 26 (anemia)

Hgb (g/dL) 16.3 14.0 8.3

RBC (106/µL) 7.6 6.5 3.6

MCV (fL) 64 67 72 (macrocytic)

MCHC (g/dL) 33.8 32.3 32.0 (normochromic)

Observations and CBC data: 14-day acute oral study of Bromadiolone in dogs

Clinical observations:Depression, vomiting and diarrhea by 5 days post-dosingBleeding at venipuncture site as early as 6 days post-dosingBleeding at various sites (i.e., urinary tract, nose, gums, subcutaneous tissue -

hematomas, G. I. tract - blood/melena) occurred as early as 8 days post-dosing

Pale mucous membranes observed at 11 days post-dosing

CBC (means) day 0 day 7 day 11Hct (%) 48.3 43.4 26 (anemia)

Hgb (g/dL) 16.3 14.0 8.3

RBC (106/µL) 7.6 6.5 3.6

MCV (fL) 64 67 72 (macrocytic)

MCHC (g/dL) 33.8 32.3 32.0 (normochromic)

Morphological Classification: macrocytic, normochromic anemiaIs it responsive?

0%

200%

400%

600%

800%

1000%

1200%

0 1 3 5 7 9 11 13

Days

% o

f C

on

trol

ACTRVVTPT

Coagulation Data for Dogs Administered Bromadiolone

(ACT, APTT) (PT)

(RVVT)COMMON SYSTEM

(~18-36 hrs)

(~35-60 hrs)

COMMON SYSTEM

(~20-40 hrs)

Circulating half-lives of the vitamin K-dependant

clotting factors

(~18-36 hrs)

(~35-60 hrs)

(~2-7 hrs)

COMMON SYSTEM

(~20-40 hrs)

Circulating half-lives of the vitamin K-dependant

clotting factors

Structure of the GPIb-IX-V receptor

Tablin, 2000

Hemostatic Platelet Functions

Thompson & Harker, 1983