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 ² 100,000 Chains alternating N-acetylglucosamine and Nglucuronic acid Purified heparin polysaccharide chains of 3,000 ² 35,000 with average molecular weight of 12,000

Pharmacokinetics of Heparin   

Intravenous administration ² immediate anticoagulation half life of one hour anticoagulant effect for 2 ² 6 hours Subcutaneous administration ² 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 ² 0.7 units/ml Specific heparin assay, inhibition of Xa measured with chromogenic substrate APTT

Establishment of Therapeutic Range for APTT 

BrillBrill-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 ² 0.7 units/ml Determined from dose response curve using regression analysis Getting enough patients Doing heparin assay 

Problems for small hospital 


Example of Brill-Edwards Method BrillSpecimen 1 2 3 4 5 6 7 8 9 10 APTT 25 45 55 88 75 65 35 47 68 98 Heparin 0.15 0.38 0.45 0.85 0.70 0.55 0.25 0.35 0.72 0.92

BrillBrill-Edwards cont . . .
APTT at 0.30 units 47 sec

APTT at 0.7 unit 75 sec Thus therapeutic range is 47 ² 75 sec approximate

Specific Heparin Assay 
 



Based on inhibition of Xa Heparin forms complex with antithrombin (heparin(heparin-AT) HeparinHeparin-At +Xa AT-Xa + Xa ATFree 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 80unfractionated 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 continuedantibody 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/mm3 or count of less that 50% of the platelet count before heparin was started Occurs 5 ² 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 radiodonor platelets Daily platelet counts recommended for all patients on heparin

Treatment of HIT ² 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 ² 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

HalfHalf-Lives of Vitamin K Dependent Factors
II VII IX X Protein C Protein S 60 Hours 4 ² 6 Hours 20 ² 24 Hours 48 ² 72 Hours 8 Hours 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 ² 3.0 for most conditions INR 2.5 ² 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 ² 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 nmfactor 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 ² 165%) 

   

Thrombin time 21.6 sec (16.4 ² 20.7 sec) Fibrinogen 642 mg/dl (174-442 mg/dl) (174Fibrinogen antigen 750 mg/dl (180 ² 310 mg/dl) Reptilase time 16.1 sec (15.5 ² 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 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 ² 20 hours, once per day dosing

Fondaparinux Used for Prophylaxis 


2.5 mg daily Peak level 0.39 ² 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 ² 100 kg body weight 7.5 mg/day Over 100 kg body weight 10.0 mg/day Anticipated peak plasma levels 1.20 ² 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 ² 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 ² 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 2.5 baseline APTT

Adverse Affects of Lepiruden 


Hemorrhage No known antidote

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