Monitoring the Traditional & the

New Anticoagulants

Michael J. Sanfelippo, M.S.

Technical Director, Coagulation Services
Marshfield Labs
 Anticoagulants to be Discussed
 Heparin
 Warfarin
 Low Molecular Weight heparin
 Fonaparienux
 Direct Thrombin Inhibitor
 Unfractionated Heparin Chemical
Composition
 Sulfated glycosaminoglycan
 Polysaccharide chains with moleculr weights
of 30,000 – 100,000
 Chains alternating N-acetylglucosamine and
glucuronic acid
 Purified heparin polysaccharide chains of
3,000 – 35,000 with average molecular weight
of 12,000
 Pharmacokinetics of Heparin
 Intravenous administration – immediate
anticoagulation half life of one hour
anticoagulant effect for 2 – 6 hours
 Subcutaneous administration – peak
anticoagulation at 4 hours with effect up to 12
hours
 In high doses, most is excreted in the urine
unaltered
 Use of Heparin
 Immediate anticoagulant
 Activity is variable requires monitoring
 Used for initial treatment of DVT and PE
 Used in open heart surgery
 Used to cap intravenous lines
 Factors Affecting Anticoagulant
Effect
 Level of Antithrombin
 Heparin binding to acute phase proteins
 Release of platelet factor 4
 Monitoring Heparin
 Therapeutic range 0.3 – 0.7 units/ml
 Specific heparin assay, inhibition of Xa
measured with chromogenic substrate
 APTT
Establishment of Therapeutic Range
for APTT
 Brill-Edwards Method
 Measure heparin level in at least 30 patients on heparin only.
 Do APTTs on all specimens
 Calculate dose response curve by regression analysis
 Therapeutic range is APTT range that corresponds to 0.3 –
0.7 units/ml
 Determined from dose response curve using regression
analysis
 Problems for small hospital
 Getting enough patients
 Doing heparin assay
Example of Brill-Edwards Method
Specimen APTT Heparin
1 25 0.15
2 45 0.38
3 55 0.45
4 88 0.85
5 75 0.70
6 65 0.55
7 35 0.25
8 47 0.35
9 68 0.72
10 98 0.92
 Brill-Edwards cont . . .
APTT at 0.30 units 47 sec

APTT at 0.7 unit 75 sec

Thus therapeutic range is 47 – 75 sec

approximate
 Specific Heparin Assay
 Based on inhibition of Xa
 Heparin forms complex with antithrombin
(heparin-AT)
 Heparin-At +Xa AT-Xa + Xa
 Free Xa + chromogenic substrate
peptide +Paranitroanaline
Heparin Assay
 Protamine Sulfate
 Low molecular weight protein from salmon
sperm
 Inhibits heparin effect by displacing heparin
from antithrombin
 1 mg protamine will neutralize 80-100 units
of unfractionated heparin
 Heparin rebound due to protamine being
cleared faster than heparin
Adverse Effects of Protamine Sulfate
 Hemorrhage
 Allergic reaction
Heparin Induced Thrombocytopenia
 Type I Platelet count drops slightly but returns
to normal while heparin is continued-not
antibody mediated
 Type II Severe antibody mediated
thrombocytopenia with life threatening arterial
and venous thrombosis if heparin is continued
Heparin Induced Thrombocytopenia
Type II
 Potentially fatal complication of heparin
therapy due to the development of a heparin
dependent antibody against platelet factor 4
 Thrombocytopenia results from activation of
platelets by complex of IgG, heparin, and
platelet factor 4
 Platelet activation causes release of ADP
which causes platelet aggregate formation
Diagnostic Features of HIT Type II
 Platelet count of less than 100,000/mm 3 or count
of less that 50% of the platelet count before
heparin was started
 Occurs 5 – 12 days after heparin is started
 Abnormal results in tests for antibody to platelet
factor 4 or heparin mediated platelet activation
 Occurrence of arterial or venous thrombus
formation while on heparin
 Tests for HIT Type II
 Elisa assay for antibody to platelet factor 4
 Release of radio-labeled serotonin from
normal donor platelets
 Daily platelet counts recommended for all
patients on heparin
 Treatment of HIT – Type II
 Stop heparin
 Use direct thrombin inhibitors
 Argatroban
 Lepiruden (Refluden®)

 Warfarin is contraindicated
Low Molecular Weight
Heparin
Low Molecular Weight Heparin
 Made by enzymatic or chemical
depolymerization
 Lower molecular weight 4,000 – 5,000
 Loss of most anti IIa activity but retain anti Xa
 Predicable anticoagulant activity
Administration and Monitoring Low
Molecular Weight Heparins
 Given subcutaneously
 Peak level at 4 hours post injection
 Half life of 4.5 hours with significant activity
at 12 hours
 Laboratory Monitoring Low
Molecular Weight Heparin
 Usually not required
 Assayed by inhibition of Xa using a
chromogenic substrate
 Same assay as unfractionated heparin but
different calibrator
 APTT not reliable but may be slightly
prolonged
Conditions Requiring Monitoring of
Low Molecular Weight Heparin
 Patients with renal insufficiency
 Exceptionally large or small patients
 Newborn and children
Low Molecular Weight Heparins in
Common Use
 Enoxaparin (Lovenox®)
 Daltaparin (Fragmin®)
 Both calibrated against the same standard
Warfarin
 Action of Warfarin
 Inhibits vitamin K epoxide reductase
 Prevents attachment of glutamic acid residues
to factors II, VII, IX, X, protein C, and protein
S
 Glutamic acid residues necessary for factor to
bind to catalytic phospholipid surfaces
 Methods of Monitoring
 Accepted method is one stage prothrombin
time
 Assay of factor X with chromogenic substrate
for special conditions
 Traditional clotting assay of factor X
Half-Lives of Vitamin K Dependent
Factors
II 60 Hours
VII 4 – 6 Hours

IX 20 – 24 Hours

X 48 – 72 Hours

Protein C 8 Hours
Protein S 30 Hours
 Monitoring Warfarin with
Prothrombin Time
 Reported as International Normalized Ration
(INR)
 Reporting as clotting time in seconds only is
not acceptable
 INR
Calculated by the formala

INR = Patient Time (sec)/Normal Mean Time(sec) ISC

Where ISC = International Sensitivity Index

ISI
Test Plasma Time (sec)
INR
Mean Normal Plasma Time
 Therapeutic Range
 INR 2.0 – 3.0 for most conditions
 INR 2.5 – 3.5 for patients with mechanical
heart valves
 Use of the Chromogenic Factor X
Assay
 Patients with the lupus anticoagulant
 Patients with abnormal fibrinogen and
problem in detection of fibrin endpoint
 Therapeutic range 43% to 17% correspondes
to INR 2.0 – 3.5
 The Assay of Factor X with a
Chromogenic Substrate
 Factor X is activated by Russell’s Viper
Venom and Ca++
 Factor Xa hydrolyses substrate (S2765)
liberating paranitroanaline
 Optical density read at 405 nm-directly related
to factor X concentration
 Effect of Lupus Anticoagulant on
Prothrombin Time
 May bind phospholipid in test reagent
 May also bind prothrombin
 Case History of Patient with
Abnormal Fibrinogen
 65 year old female with atrial fibrillation on
warfarin for several years with good control
 Had INR performed at a different lab with a
value of > 11.0
 Warfarin stopped
 Warfarin restarted with INRs at original lab
acceptable
 INR performed at different lab with > 11.0
again
 Case History cont . . .
 Duplicate specimens drawn
 Original lab INR 1.3
 Other lab INR > 11.0
 Chromogenic X 79% (Normal 65 – 165%)
 Thrombin time 21.6 sec (16.4 – 20.7 sec)
 Fibrinogen 642 mg/dl (174-442 mg/dl)
 Fibrinogen antigen 750 mg/dl (180 – 310 mg/dl)
 Reptilase time 16.1 sec (15.5 – 19.0 sec)
 Similar to Fibrinogen Longmont
 Adverse Affects of Warfarin
 Bleeding, anticoagulation reversal by vitamin
K
 Tissue necrosis
 Occurs in patients with underlying protein C or S
deficiency prevented by giving small loading dose
of 2 – 5 mg/day starting warfarin while patient is
on heparin
Effect of Unfractionated Heparin on
INR
 Heparin can prolong the prothrombin time
(increase INR)
 Most reagents now contain a heparin
antagonist protamine sulfate or polybrene
 Most reagents will neutralize up to 1.0
units/ml
Fondaparinux
 Chemistry and Action of
Fondaparinux
 A pentasaccharide with a molecular weight of
1,728 daltons
 Anticoagulates by accelerating the binding of
antithrombin to Xa
 Has no effect on thrombin binding
 Therapeutic Levels for
Fondaparinux
 Administered by subcutaneous injections
 Peak level at 3 hour post injection
 Half life 14 – 20 hours, once per day dosing
Fondaparinux Used for Prophylaxis

 2.5 mg daily
 Peak level 0.39 – 0.50 mg/L
 Fondaparinux Used for Treatment
of Deep Venous Thrombosis or
Pulmonary Embolism

 50 kg body weight 5.0 mg once/day

 50 – 100 kg body weight 7.5 mg/day
 Over 100 kg body weight 10.0 mg/day
 Anticipated peak plasma levels 1.20 – 1.26
mg/L
 Methods of Monitoring
 Common laboratory tests, prothrombin time and
activated partial thromboplastin time are insensitive
to fondaparinux
 Specific assay based on inhibition of Xa with
calibration curve using fondaparinux
 Monitoring generally not required due to the
predictability of the drugs anticoagulant action
 Should be monitored in patients with renal
impairment and elderly patients (over 75 years)
 Should be monitored in patients less than 50 kg body
weight
 Advantages of Fondaparinux
 Long half life
 Low incidence of HIT, may be used to treat
patient who have suffered HIT
 Generally doesn’t require monitoring
 Argatroban ®

 A small molecule molecular weight of 526

 Derived from arginine
 Inhibits by reversibly binding to thrombin
independent of antithrombin
 Half life of 39 – 51 minutes
Therapeutic Levels and Monitoring
 Given by continuous infusion
 Monitor with activated partial thromboplastin time
(APTT)
 At 2 hour of infusion target APTT range is 1.5 – 3.0
times baseline
 Argatroban® will prolong prothrombin time (PT)
 Use chromogenic assay factor X to monitor warfarin
during transition from argatroban to warfarin
 Drug Use and Adverse Affects
 Primary use to treat patients with HIT
 Hemorrhage is the only major adverse affect
 No known antidote
Lepiruden (Refluden )
®
Therapeutic Levels and Monitoring
 Monitored by APTT
 General target range 1.5 – 2.5 baseline APTT
 Adverse Affects of Lepiruden
 Hemorrhage
 No known antidote