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.
Platelet - TEM
microtublules
OCS
granules
mitochodrion
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 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).
Platelet Response to Agonists
Platelets - unstimulated
Characteristic discoid shape
SEM plates;
Gentry, 2000
Addition of ADP
Addition of thrombin
(mild stimulation)
(strong stimulation )
Shape change (elongation
and crescents) and filaform
process formation (arrows)
Increased spreading,
filaform process extension
(arrows) and aggregate
formation (stars)
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
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 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, a2macroglobulin, 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).
Coagulation Inhibitors
Coagulation is controlled by three types of actions.
• Inhibition of converting enzymes (e.g., AT III, C1 esterase inhibitor, a2macroglobulin, 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).
Proposed Mechanism of AT III-Heparin
System
Lysine
sites
Serine site
AT III
Arginine
site
Thrombin
Antithrombin
III
Th
Heparin
H
AT III
Th
H
Coagulation Inhibitors
Coagulation is controlled by three types of actions.
• Inhibition of converting enzymes (e.g., AT III, C1 esterase inhibitor, a2macroglobulin, 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).
Proposed Mechanism of Thrombomodulin,
Protein C and Protein S (TM-PC-PS) System
F-Xa
Prothrombin
Activated
platelet
PS
Ca++
F-Va
Thrombin
Activated PC associates with PS and degrades cofactors V and VIII:C
Ca++
Activated
Protein C
Thrombin
Thrombomodulin
Protein C
Coagulation Inhibitors
Coagulation is controlled by three types of actions.
• Inhibition of converting enzymes (e.g., AT III, C1 esterase inhibitor, a2macroglobulin, 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).
Proposed Mechanism of Tissue Factor
Pathway Inhibitor (TFPI) Activity
F-Xa
F-Xa
TFPI
TFPI
F-Xa
TFPI
F-VIIa Tissue factor
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.
Fibrinolytic System and Factors Regulating
Fibrinolysis (Fibrinogenolysis)
Activation
Damaged
endothelium
Plasminogen
Inhibition
Kallikrein
Plasminogen activator inhibitor
e-aminocaproic acid
FHIIa
Prekallikrein
tPA
Streptokinase
Urokinase
Plasmin
a2-Antiplasmin
Biodegradation of
FV, FVIII, FIX, FXI
fibrinogen
Firbrinogen/fibrin
a2-Macroglobulin
Complement activation
Fibrin/fibrinogen
Degradation products
Degradation of Fibrin/Fibrinogen
Fibrinogen or Fibrin
Plasmin
Fragment X
Small Peptides
Plasmin
Fragment Y
Fragment D
Small Peptides
Plasmin
Fragment E
Fragment D
Small Peptides
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
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 Anticoagulants
Acute (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
Precoagulant
Clotting Factors
(II, VII, IX, X)
Functional
Clotting Factors
(II, VII, IX, X)
CO2
Vitamin K
reduced
Vitamin K
epoxide
X
Vitamin K epoxide reductase
In hepatocytes
(Coumarins inhibit this enzyme)
COMMON SYSTEM
(ACT, APTT)
(PT)
COMMON SYSTEM (RVVT)
(ACT, APTT)
(PT)
COMMON SYSTEM (RVVT)
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-dosing
Bleeding 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-dosing
Bleeding at venipuncture site as early as 6 days post-dosing
Bleeding 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-dosing
Bleeding at venipuncture site as early as 6 days post-dosing
Bleeding 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-dosing
Bleeding at venipuncture site as early as 6 days post-dosing
Bleeding 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)
Hct (%)
Hgb (g/dL)
RBC (106/µL)
MCV (fL)
MCHC (g/dL)
day 0
day 7
day 11
48.3
16.3
7.6
64
33.8
43.4
14.0
6.5
67
32.3
26
8.3
3.6
72
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-dosing
Bleeding at venipuncture site as early as 6 days post-dosing
Bleeding 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)
Hct (%)
Hgb (g/dL)
RBC (106/µL)
MCV (fL)
MCHC (g/dL)
day 0
day 7
day 11
48.3
16.3
7.6
64
33.8
43.4
14.0
6.5
67
32.3
26 (anemia)
8.3
3.6
72
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-dosing
Bleeding at venipuncture site as early as 6 days post-dosing
Bleeding 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)
Hct (%)
Hgb (g/dL)
RBC (106/µL)
MCV (fL)
MCHC (g/dL)
day 0
day 7
day 11
48.3
16.3
7.6
64
33.8
43.4
14.0
6.5
67
32.3
26 (anemia)
8.3
3.6
72 (macrocytic)
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-dosing
Bleeding at venipuncture site as early as 6 days post-dosing
Bleeding 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)
Hct (%)
Hgb (g/dL)
RBC (106/µL)
MCV (fL)
MCHC (g/dL)
day 0
day 7
day 11
48.3
16.3
7.6
64
33.8
43.4
14.0
6.5
67
32.3
26 (anemia)
8.3
3.6
72 (macrocytic)
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-dosing
Bleeding at venipuncture site as early as 6 days post-dosing
Bleeding 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 11
Hct (%)
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 anemia
Is it responsive?
Coagulation Data for Dogs Administered Bromadiolone
1200%
ACT
RVVT
PT
% of Control
1000%
800%
600%
400%
200%
0%
0
1
3
5
7
Days
9
11
13
(ACT, APTT)
(PT)
COMMON SYSTEM (RVVT)
Circulating half-lives of the
vitamin K-dependant
clotting factors
(~18-36 hrs)
COMMON SYSTEM
(~20-40 hrs)
(~35-60 hrs)
Circulating half-lives of the
vitamin K-dependant
clotting factors
(~18-36 hrs)
(~2-7 hrs)
COMMON SYSTEM
(~20-40 hrs)
(~35-60 hrs)
Structure of the GPIb-IX-V receptor
Tablin, 2000
Hemostatic Platelet Functions
Thompson &
Harker, 1983