Overview of Hemostasis

Understanding Blood
Vessel
Anticoagulants Injury
Contact/
Neural
Tissue
Mechanism Platelet Factor
Activation

The Good . . . and the Bad

Blood Vessel Platelet Coagulation
Constriction Aggregation Cascade
Primary hemostatic plug

Alan P. Agins, Ph.D. Reduced

Fibrin
Blood
President, PRN Associates formation
flow
Continuing Medical Education, Tucson, AZ

Stable Hemostatic Plug

The Platelet Aggregation Step Fibrinogen

Fibrinogen

Thrombin Thromboxane A2 Thrombin Thromboxane A2

Ca++ Ca++
ADP ADP
Ca++ Ca++

Glycoproteins
Ilb, IIIa
Glycoproteins Exposed Collagen

ct e
F a ssu
Ilb, IIIa

or
VWF VWF

Ti
Endothelium
Exposed Collagen

Glycoproteins
cyclooxygenase Ilb, IIIa ADP
AA TxA2 TxA2

Collagen Collagen
VWF VWF

Exposed Collagen Exposed Collagen

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 The exciting, but really complicated
biochemistry involving “factors” steps
Exposed Phospholipid Surface site
for coagulation cascade activity
Intrinsic Ti
Fa ssu
Pathway ct e
or
Contact
TxA2 ADP
Activation
Pathway

Collagen
VWF Extrinsic
Pathway

Exposed Collagen

Intrinsic Extrinsic

Final Common Checks and Balances

Pathway • Five mechanisms keep platelet activation and
the coagulation cascade in check.
• Abnormalities can lead to an increased
tendency toward thrombosis:
– Protein C
– Antithrombin
– Plasmin
– Tissue factor pathway inhibitor
– Prostacyclin

Hemostasis Causes of Thrombosis

Hemostatic plug Endothelial cell
Platelets Fibrin RBC • Composition of the blood
(hypercoagulability)
• Quality of the vessel wall
(endothelial cell injury)
Virchow's
• Nature of the blood flow
triad
(hemostasis)

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Arterial Thrombus Venous thrombus
Usually occur in association with Usually in the lower limbs
pre-existing vascular disease,
the most common of which is
- often asymptomatic
atherosclerosis ~ plaque rupture Can produce acute symptoms if they cause
Produce clinical manifestations inflammation of the vessel wall, obstruct flow,
by inducing tissue ischemia, or embolize into the pulmonary circulation
either by obstructing flow or by
embolizing into the distal
microcirculation

May lead to MI, occlusive stroke

or other ischemic events

Risks for Thrombosis Indications For Anticoagulant Therapy

CAD / plaque rupture
Recent Surgery
Immobilization causing stasis of blood.
Travel
Obesity
Malignancy, especially adenocarcinoma
Previous history of DVT or pulmonary
embolus (PE)
Pregnancy (up to 2 months postpartum)
Fracture
Heart failure (causes stasis)
Oral contraceptive / Estrogen use
Arterial thromboembolic disease
• Prosthetic heart valves
• Mitral valve disease, especially with
atrial fibrillation
• Congestive cardiomyopathies,
especially with AF
• Atrial fibrillation
• Mural cardiac thrombi
• Transient ischemic attacks
• Stroke in evolution

Indications For Anticoagulant Therapy Heparin

Venous thromboembolic disease Mucopolysaccharide: MW from 6,000 to 40,000 Da.
• Deep venous thrombosis (DVT) Average MW of commercial preps: 12,000 - 15,000.
• Pulmonary embolism (PE)
• Primary prophylaxis of DVT or PE Key structural unit of heparin is a unique
pentasaccharide sequence
• Disseminated intravascular coagulation
• Maintenance of patency of vascular
grafts, shunts, bypasses

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Heparin Mechanism
Heparin’s Limitations
The Heparin : Antithrombin complex
can only bind to and inhibit soluble
thrombin – NOT firbrin-bound thrombin
Heparin Monitoring
• Activated partial thromboplastin time (aPTT)
• Termed "partial" due to the absence of tissue
factor from the reaction mixture.
• Indicator of the efficacy of both the "intrinsic"
(contact activation pathway) and the common
coagulation pathways.
• Adequate therapeutic effect = aPTT ratio of 2.0-
2.5 times of the baseline aPTT
• Monitor aPTT every 4 hours until therapeutic
range has been achieved.
• Thereafter, monitor aPTT and platelet count
daily.
Heparin’s Limitations
Binds to plasma proteins – variable bioavailability
Heparin - Adverse Effects
• Hemorrhagic events
– Antidote = Protamine Sulfate
• Non-hemorrhagic side-effects
– Elevation of serum aminotransferase levels
• reported in as many as 80% of patients
receiving heparin
• not associated with liver dysfunction
• disappears after the drug is discontinued.
– Hyperkalemia
• 5 to 10% of patients receiving heparin
• Due to aldosterone suppression.
• Can appear within a few days
• Rare side-effects- alopecia and osteoporosis
Heparin Monitoring
• Anti-Xa levels may be more reliable than aPTTs
for monitoring heparin in newborns and some
children:
• At birth, aPTT is prolonged, reflecting the
immaturity of the coagulation system.
• Children requiring heparin therapy frequently
have underlying disorders that influence the
baseline aPTT and therefore the response to
heparin.
• Elevated factor VIII may cause subtherapeutic
aPTT despite adequate anti-Xa level.
• In these children, aPTT may not correlate well
with anti-Xa levels and thus checking both
parameters may be helpful. In such conditions,
anti-Xa should be used.
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Low Molecular Weight Heparin
• LMWH: 4000-5000 (vs Heparin: 15,000+)
LMWHs inactivate Xa but have less effect on thrombin
(some molecules not long enough)
– ratio of anti-Xa to anti-thrombin activity of 3:1
– Do not prolong PTT unless dose high
• Advantages over heparin:
– Easier to administer: sc, BID dosing
– Dosage and anticoagulant effect easier to predict;
dose based on body weight
– Lab monitoring not necessary in all patients
– Less chance of inducing immune-mediated
thrombocytopenia
– Smaller risk of osteoporosis in long-term use

Names of LMWHs LMWH Rx monitoring

• Enoxaparin (Lovenox) • Uncomplicated patients do not require
• Dalteparin (Fragmin) monitoring
• Tinzaparin (Innohep) • Who may need to be?
– Differ chemically and pharmacokenetically – Newborns, children, pregnant women
but unsure if these differences are clinically – Conditions: obesity, renal insufficiency,
significant malignancy, myeloproliferative disorders
• Other products not yet approved here: – Pts with hemorrhagic complications or with
– Fraxiparin, reviparin, nadroparin, initial therapy to confirm appropriate levels
bemiparin, certoparin
• Anti-Xa for monitoring

Why do we need newer

Categories of New Drugs
Anticoagulants?
• UFH and LMWHs are inconvenient for –Factor Xa inhibitors:
the outpatient setting (IV or sq only) • fondaparinux, idraparinux
–Direct Thrombin Inhibitors:
• UFH and LMWHs can cause HIT: • hirudin, lepirudin, desirudin,
– Risk 0.2% with LMWH vs. 2.6 % with UFH bivalirudin, argatroban,
– Pts with HIT still need to be anticoagulated ximelagatran
–Heparinoids:
• Danaparoid (discontinued)

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Factor Xa inhibitors Fondaparinux (Arixtra)
Intrinsic Extrinsic
• Fondaparinux (Arixtra) pathway pathway

– Synthetic polysaccharide: Antithrombin

– The drug is the unique pentasaccharide sequence that
UFH and LMWH use to bind to AT ATIII ATIII ATIII Xa Xa

Fondaparinux
II IIa

Too short to inactivate thrombin (much like LMWH); need >18

saccharide units to inactivate thrombin Fibrinogen Fibrin
– Reacts with strong affinity to AT (reversible) → clot

Induces conformational change in AT → Platelets IIA

Increased ability to inactivate Xa

Fondaparinux Fondaparinux
– Does not interact with plasma proteins, • Drug monitoring:
platelets, or platelet factor IV = useful in HIT • APTT and PT are insensitive
(although not yet formally approved) • PT/INR may or may not be proportional to
– FDA approved in 2001 the clinical safety or efficacy—more studies
• Prevention of post op VTE (DVT and PE) in needed
orthopedic surgery • Anti-factor Xa assay –must be calibrated
– Hip fracture, hip replacement, knee replacement
with fondaparinux
– Fondaparinux vs. enoxaparin in one study decreased
VTE in knee replacement from 12.5 to 27.8% – Long half-life (17 hours) = qd dosing
• 2004/5 approval: (LMWH = BID)
– VTE treatment if administered with warfarin
– Anticoagulation in abdominal surgery Idraparinux
• Potential uses being studied: MI, PCI, UA – Longer acting analogue (q week dosing)
currently being developed

Comparison Parenteral Anticoagulants

Factor Xa inhibitors - Summary
• Alternative agent for LMWH for
prophylaxis or initial treatment of
venous thromboembolism
• Treatment of HIT
• No true antidote although Factor VIIa
may be of benefit
• Ongoing trials with Idraparinux

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Direct Thrombin Inhibitors Direct Thrombin Inhibitors
Heparin binding site
• Two types
IIa
– Bivalent DTIs (hirudin and analogs)
– Univalent DTIs bind only to the active site
Hirudins
• Direct thrombin inhibitors block both Catalytic IIa
circulating thrombin and clot-bound thrombin. site
• There is no therapeutic drug monitoring
widely available for DTIs
Argatroban
• The ecarin clotting time, although not in Substrate Melagatran
IIa
general clinical use, would be the most Recognition
appropriate monitoring test. Site

Bivalent DTIs IIa

Univalent DTIs
IIa
Ximelagatran
• All bind in active site and exosite I – First oral direct thrombin inhibitor
– Prodrug of melagatran
• IV, IM, SC
– Discontinued (2006) due to Liver Toxicity
• Reversible: Bivalirudin Argatroban
• Irreversible: Lepirudin, Desirudin – IV
– Second agent (the first is lepirudin) to be indicated
• Minor differences in structure for heparin-induced thrombocytopenia (HIT).
• Approved for acute coronary syndrome ("unstable – Hepatically eliminated and can be used in patients
angina") with end-stage renal disease.
• Less suitable for long-term treatment

Advantages of direct thrombin inhibitors Oral Anticoagulants

No nonspecific binding to Predictable anticoagulant
plasma proteins response

Not neutralized by platelet Retains activity in presence of

factor 4 (PF4) platelet-rich thrombi

Ability to inactivate free and Completely inhibits fluid-phase

bound thrombin and fibrin-bound thrombin

Inhibits thrombin-mediated No activation of clotting cascade

platelet activation or release of binding proteins

No formation of heparin-PF4 No heparin-induced

complexes thrombocytopenia

Courtesy of R Mehran, MD.

Warfarin

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WARFARIN Warfarin: Mechanism of Action
Serendipitous history
“WARF” = Wisconsin Alumni Research Foundation Vitamin K
(the holder of original patent)
Taken by six – seven million patients in United States
VII Synthesis
THERAPEUTIC USES: IX of Non-
Prophylaxis and treatment of venous thrombosis Functional
X Coagulation
Treatment of atrial fibrillation with embolism II Factors
Prophylaxis & treatment of pulmonary embolism
Warfarin
Adjunctive therapy for coronary occlusion

Prophylaxis in patients with prosthetic valves

Warfarin

• Racemic mixture of two active optical isomers -

R & S forms
• Both isomers active, however S-warfarin has
5X the potency of the R-isomer and modulates
the in vivo activity of warfarin.
• Each isomer is cleared by different cytochrome
P450 pathways.
• CYP2C9 is the principle enzyme that
metabolizes S-warfarin
• CYP1A2 & CYP3A4 metabolize the R-isomer.

Warfarin - Adverse Effects Warfarin Monitoring

“narrow therapeutic index” Prothrombin Time
ƒ Hemorrhage • The time it takes plasma to clot after
Risk of severe bleeding small (1-2%/yr) but definite
Any benefit needs to outweigh this risk when addition of tissue factor
warfarin is considered as a therapeutic measure. • Measures the extrinsic pathway and
ƒ Skin necrosis final common pathway (factors II, V,
ƒ Purple toe syndrome / Cholesterol embolism VII, X and fibrinogen)
ƒ Teratogenecity • An estimated 800 million PT/INR assays
ƒ Osteoporosis are performed annually worldwide
ƒ Agranulocytosis, leukopenia, diarrhea,
nausea, anorexia

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Warfarin Monitoring Desired Therapeutic Range
Indication INR
Terms: Prophylaxis of venous 2.0-3.0
PT ratio (PTR) = Patient’s PT thromboembolism
Treatment of venous 2.0-3.0
Control PT thromboembolism
ISI - International Sensitivity Index Atrial fibrillation 2.0-3.0
(standard for thromboplastin reagent) Mitral valve stenosis 2.0-3.0
Heart valve replacement
INR = PTRISI Bioprosthetic valve
Mechanical valve
2.0-3.0
2.5-3.5
normal range for the INR is 0.8-1.2
Myocardial infarction 2.0-3.0
2.5-3.5 (high risk patients)

Timing of INR Monitoring Genetic Variability

Coagulation factors t1/2 vary from 6 – 72 hours Vitamin K epoxide reductase
T1/2 of warfarin ranges from 1- 2.5 days

1st INR – 2 to 3 days – then daily until therapeutic for
at least 2 consecutive days
Followed by - INR 2-3 times weekly for 1 – 2 weeks
Gradually reduce frequency to q4weeks (if stable)
Remember
Changes made in Warfarin dose are not completely
reflected in the INR until day 3 or 4
• Polymorphisms in the vitamin K epoxide
reductase complex 1 (VKORC1) gene explain
30% of the dose variation between patients
• The Good: Polymorphisms lead to a more
rapid achievement of a therapeutic INR
• The Not-so-Good: Shorter time to reach an
INR > 4, which is associated with bleeding
• African-Americans are relatively less sensitive
to warfarin
• Asian Americans are more sensitive

Genetic Variability Warfarin Interaction

Cytochrome P450 9 Alter metabolic clearance
Genetic polymorphism of CYP2C9 may play a role 9 Reduce absorption from the intestine
in the interpatient variability of response to warfarin
9 Inhibit synthesis of vitamin K-dependent
and predisposition to drug interactions.
coagulation factors
Polymorphism of CYP2C9 exists in approx 10% of 9 Increase metabolic clearance of vitamin K-
Caucasians (very rare in African American or Asian populations) dependent coagulation factor
CYP2C9 polymorphisms do not influence time to 9 Interfere with other pathways of hemostasis
reach effective INR (as opposed to VKORC1) but
9 Unknown mechanisms
do shorten the time to INR >4

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Drugs / Lifestyle / Dietary Interaction Drugs / Lifestyle / Dietary Interaction
Agents that may reduce the effectiveness of oral Increase hemorrhage risk by inhibiting
anticoagulants – Increase risk of Thrombosis metabolic clearance of warfarin (increase INR)
CYP 1A, 2C, 3A
Increase Metabolic Reduce Absorption
Clearance – Bile Acid Resins Omeprazole Erythromycin
– Carbamazepine Direct Antagonism
Amiodarone Clarithromycin
– Rifampin – Vitamin K (dietary)
Ketoconazole Fluoroquinolones
Itraconazole Protease Inhibitors
– Phenytoin
Independent Risk Factors Fluconazole Grapefruit juice
– St John’s wort
– Oral contraceptives Metronidazole
– Cigarette smoking
– Estrogen / SERMs Cimetidine Many others
– Cruciferous
vegetables

Drugs / Lifestyle / Dietary Interaction Risks Related to Anticoagulants

Increase hemorrhage risk by other mechanisms
(pharmodynamic, pharmacokinetic, multiple pathways, etc)
• Antiplatelet Drugs
• Botanical supplement affecting platelets
• Ginger, ginkgo, garlic, feverfew, St John’s wort
• Glucosamine / chondroitin supplements (increases INR)
• Alcohol
• Some cephalosporin and sulfa antibiotics
• COX-2 inhibitors
• Valproate
• Many others
• Warfarin has a narrow therapeutic index
• Between 1993 & 2006 ~ 9,766 reports of bleeding
complications related to warfarin therapy.
– 86% of these were considered serious / 10% were fatal.
• In April 2006, Warfarin ranked ninth in the list of
drugs with the most reported adverse events
(4,861 cases).
• In October 2006, a black box warning of the risk of
bleeding was added to the package inserts for
proprietary and generic warfarin products in the
U.S.

Education is the key to

keeping patients safe Antiplatelet
– Proper dosing Drugs
– Compliance
– Potential for interactions with other drugs,
foods, lifestyle, etc
– Need for routine bloodwork
– Plan for handling bleeding
• Minor
• Major

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 ADP
receptor As TxA2 (Thromboxane)
pir
in Why Low Dose ASA? Increases calcium flux
Clopidogrel
• Adhesion
Ticlopidine
• Aggregation
• Vasoconstriction

Dipyradimole PGI2 (Prostacyclin)

ReoPro
Ca++ cyclooxygenase Increases cAMP
AA Aspirin TxA2 Aggrestat
Integralin
AA
X
cyclooxygenase
TxA2 • Prevents Adhesion
• Prevents Aggregation
Ca++ • Vasodilation

Collagen
VWF

cyclooxygenase
Endothelium AA
X PGI2

TxA2 (Thromboxane)
Why Low Dose ASA? Increases calcium flux Aspirin
• Adhesion
• Aggregation
• Vasoconstriction – Antiagregation occurs within 1hr
– Side Effects: Allergy, GI discomfort, GI bleed
No nucleus PGI2 (Prostacyclin)
Increases cAMP
– OTC, easy to crush, inexpensive, small tablet,
No new
AA
X
cyclooxygenase
protein synthesis
TxA2
(ie., cyclooxygenase) for
• Prevents Adhesion
• Prevents Aggregation
enteric coated available to decrease stomach
upset
life of platelet • Vasodilation – Remember: Aspirin’s effect on
cyclooxygenase is irreversible. It will take at
least 7 - 8 days after stopping therapy to
completely restore platelet function
Nucleated Cells
cyclooxygenase
Endothelium AA
Produce mRNA
X PGI2
Synthesize new cyclooxygenase
Continue to make beneficial PGI2

ADP
receptor
Clopidogrel
Clopidogrel
Ticlopidine
– Takes 2 to 3days; maximum inhibition between 4
and 6 days
– Oral loading dose of 300mg results in faster
platelet inhibition (2-3hrs)
Ca++ cyclooxygenase
AA TxA2 – Alternative in ASA allergy / aspirin resistance
– Fewer gastrointestinal hemorrhages than aspirin,
Ca++ but more diarrhea and rash
Collagen
– Once daily, prescription required, expensive
VWF – Drug requires conversion to active metabolite
(CYP 3A4)
– Like ASA, effect is irreversible. It will take at least
7 - 8 days after stopping therapy to completely
restore platelet function

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 Antiplatelet Therapy: Common Oral Agents
Ticlopidine
– MOA not entirely known (similar to clopidogrel) Acetylsalicylic Clopidogrel Ticlopidine
acid (ASA)
– Takes 2 to 3days; maximum inhibition between
Class Salicylate Thienopyridine Thienopyridine
4 and 6 days
– many adverse events (diarrhea 20%, other GI Formulation Active Drug Pro-Drug Active Drug

sx, rash, neutropenia is rare 1% but severe – Maintenance 75-325 mg daily 75 mg daily 250 mg twice daily
occurs in first 2-3mos, also thrombocytopenic Dose
Major Bleeding 2-3% 1- 4% alone 1% alone
purpura) Risk (%) 3-5% w/ ASA 2-6% w/ ASA
– Due to serious and common side effects, and
the fact that it’s not much better than
alternatives, it’s now rarely used

ADP
receptor
Dipyridamole + Aspirin

– Increases cAMP = decreased calcium

Dipyradimole
entry = decreases platelet activation
Ca++ cyclooxygenase – 38% risk reduction for combo, better than
AA TxA2
either agent alone
– Headache is a common SE; similar
Ca++
bleeding to ASA
Collagen
– Twice daily, capsule may be opened up &
VWF
tablet crushed but granules must not be
crushed – issues with tubes, ASA allergy

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