discovery of vitamin k antagonists ( vkas ) in anticoagulation occurred in the 1920s , when the veterinarian frank schofield studied the hemorrhagic disease affecting cattle consuming sweet clover .
karl link and his team studied spoiled sweet clover and , in 1939 , extracted dicumarol and identified the hemorrhagic agent , which was the substance in the sweet clover affecting coagulation .
the discovery of dicoumarol made it possible to inhibit thrombosis and to study anticoagulation therapy for humans in the early 1940s . in the decades
1930 and 1940 professor armand quick developed a routine prothrombin time ( pt ) coagulation test , which preceded the use of vkas . this has served as a basis for oral anticoagulant therapy ( oat ) monitoring from the onset .
however this first drug for oat subsequently was found to have drawbacks by reason of its long half - life .
karl link synthesized more than 150 anticoagulant compounds and found one particularly active molecule , which was named warfarin ( a 4-hydroxy compound ) after the patent holder , the wisconsin alumni research foundation .
professor paul owren found factor v and developed a new pt method subsequent to the  quick method  to overcome its drawbacks .
the owren reagent ( combined thromboplastin reagent , thrombotest ) is used mainly in the nordic countries , benelux , and japan . by reason of its different reagent composition , the quick technique is sensitive to the coagulation factors fibrinogen , ii , v , vii , and x , while the owren technique is affected by deficiencies in factors ii , vii , and x. the quick method measures factor v and fibrinogen , which are not dependent on warfarin therapy , and this constitutes a drawback for oat .
warfarin is the most widely used drug for oat world - wide , and it has a predictable onset and duration of action and excellent bioavailability .
the therapy is effective for a variety of clinical indications of anticoagulation . the dosage and the clinical response still vary markedly among patients , depending on genetic inheritance , age , and metabolism .
warfarin is a racemic mixture of two optically active isomers , the r and s forms .
it is rapidly absorbed from the gastrointestinal tract ( within 90 min ) and its half - life is 3642 h. in the circulation warfarin is bound to plasma proteins ( mainly albumin ) .
warfarin interferes with the cyclic intercon - version of vitamin k and its 2,3 epoxide ( vitamin k epoxide ) and thus is a vka .
vitamin k is a cofactor in the carboxylation of vitamin k - dependent coagulation factors .
vka inhibits the synthesis of coagulation factors ii , vii , ix , and x in the liver , and they remain partially inactive unless 9 to 13 of the amino terminal glutamate ( glu ) residues are carboxylated to form the ca - binding -carboxyglutamate ( gla ) residues ( figure 1 ) .
this carboxylation step renders the coagulation factors functionally active : binding to ca and the phospholipid surface .
therapeutic dosages of warfarin decrease , by 3050% , the total amount of each vitamin k - dependent coagulation factor synthesized by the liver .
the secreted molecules from the liver are under - carboxylated , resulting in diminished biological activity ( 1040% of normal ) .
figure 1the enzyme reactions involved in the metabolic function of vitamin k. vitamin k - dependent carboxylase catalyzes the transformation of peptide- ( factors ii , vii , ix , and x ) bound glutamate residues ( glu ) to -carboxyglutamate ( gla ) residues in the presence of vitamin k hydroquinone , carbon dioxide , and molecular oxygen ( i ) . vitamin k hydroquinone is oxidized in the reaction to vitamin k 2,3 epoxide .
the reduction of the latter to vitamin k quinone is catalyzed by vitamin k epoxide reductase , which can use certain dithiols as the reductants ( ii ) .
vitamin k quinone can be reduced to vitamin k hydroquinone in the reactions catalyzed by either a dithiol - dependent ( iii ) or an nad(h)p - dependent ( iv ) entzyme .
( uotila l. recent findings on the functions and requirements of vitamin k in humans .
klinlab 1998 ; 3 : 97101 ) the enzyme reactions involved in the metabolic function of vitamin k. vitamin k - dependent carboxylase catalyzes the transformation of peptide- ( factors ii , vii , ix , and x ) bound glutamate residues ( glu ) to -carboxyglutamate ( gla ) residues in the presence of vitamin k hydroquinone , carbon dioxide , and molecular oxygen ( i ) . vitamin k hydroquinone is oxidized in the reaction to vitamin k 2,3 epoxide .
the reduction of the latter to vitamin k quinone is catalyzed by vitamin k epoxide reductase , which can use certain dithiols as the reductants ( ii ) .
vitamin k quinone can be reduced to vitamin k hydroquinone in the reactions catalyzed by either a dithiol - dependent ( iii ) or an nad(h)p - dependent ( iv ) entzyme .
( uotila l. recent findings on the functions and requirements of vitamin k in humans .
klinlab 1998 ; 3 : 97101 ) warfarin treatment reduces the number of gla residues ( normal , 9 to 13 ) per clotting factor molecule , with a concomitant fall in coagulant activity .
when the number of residues decreases from 13 to 9 , only 70% of the activity of the clotting factor remains ; when one molecule contains six carboxylated residues , only 2% activity is present .
the liver excretes both active and inactive coagulation factors to the plasma and both affect international normalized ratio ( inr ) measurement , which possibly has escaped notice in the world health organization ( who ) recommendation for the prothrombin time methodology .
the calculation formula is for : inr = ( samplesec / normalsec ) , where isi is the international sensitivity index . when the isi is near 1.0 , the reagent is sensitive and isi has little meaning in the inr calculation .
the quick and owren pt methods are the most common and generally accepted means of monitoring vka therapy .
a comparison between the pt methods has been published in a recent article and the owren pt was superior . who recommends the use of the inr to harmonize pt results and therapeutic ranges globally both for patient care in clinical practice and in the scientific literature , as the units used formerly proved inadequate for international communication .
the challenge is for global clinical laboratories to harmonize inr testing further to a level where inr results are consistent regardless of the methods used .
the use of the inr system still involves difficulties with sample citrate concentration , different reagents and thromboplastins , and instruments , and with isi and  local isi  calibration to harmonize results .
horsti and colleagues measured 150 samples from patients on oral anticoagulation using seven commercial reagents and four different calibrator kits .
the reagent manufacturer informs the isi value or the laboratory can measure the isi with an isi calibration kit ( local calibration ) for a pt reagent lot .
the meaning of isi as power in the inr calculation equation is displayed in the previous section .
first , the aim in the original recommendation was to harmonize inr results by calibrating reagent isi values with human combined , which was the primary reference preparation ( irp code 67/40 ) .
thromboplastins from different sources ( human brain , rabbit brain , rabbit lung , and ox brain ) yield quite different levels of pt .
the hierarchy of reference thromboplastin preparations was presented by van den besselaar and associates . as the who calibration procedure was complex and demanding in the second stage , the recommendation for isi calibration was local calibration using certified lyophilized plasmas .
this alternative means of determining the isi involves the use of freshly pooled plasmas from 20 normal individuals and 60 patients receiving coumarin ( oat ) .
such numbers of samples are necessary to obtain a precise calibration line for isi calculation . freshly pooled plasma can be used to determine reagent or instrument isi with acceptable precision , or as good a result as with the who calibration model .
poller and colleagues ( european concerted action on anticoagulation , ecaa ) have compared local isi calibration and  direct inr  in the correction for locally reported inrs . for local inr correction ( harmonization )
it is clear that plasmas from patients on warfarin therapy always involve varying amounts of inactive coagulation factors , depending on the level of anticoagulation and individual patient characteristics ( see section on warfarin ) .
all these former calibration models involve the principle that the calibrator contains an average amount of inactive coagulation factors ( inhibition ) , which means an average correction for patient inr results .
is the average correction of inactive coagulation factors appropriate for individual patient samples in a measuring range from 1 to 5 inr ?
is it a fact that calibrators , according to biochemical principles , contain inactive coagulation factors ( inhibition ) ?
do different reagents behave identically to active and inactive coagulation factors ? in our earlier studies we measured inactive coagulation factors ( inhibition ) in four different kits and noted conspicuous variation in inhibition .
isi calibration should be based on normal plasma and normal plasma dilutions without inactive coagulation factors . in principle
today the quick and owren pt methods measure the sum of active coagulation factors ( fii , fvii , fx ) and inhibition by their inactive coagulation factor counterparts .
the new - generation pt method can measure separately both active coagulation factors ( fii , fvii , fx ) and the inhibition caused by inactive or partially inactive coagulation factors .
two measurements for one sample are needed and the cost per sample is twice as much .
in addition , the number of inactive coagulation factors varies markedly in different calibrator kits ( manufactured or local ) .
the harmonization of inr results using different pt methods and reagents is problematic if attempted according to the who recommendation .
new medications for oat have been developed over a number of years and anticipated without laboratory test control in an effort to displace warfarin medication .
the new molecules are too expensive for global use or involve serious side - effects and possibly will never be as popular as warfarin , which is an old and cheap means widely used and accepted globally . the only drawback with warfarin medication is regular laboratory control
. it would be important to develop warfarin therapy with greater attention to laboratory control , which helps patient care .
different reagents and thromboplastins react variably with inactive coagulation factors and cause difficulties in calibration using patient plasmas that contain inhibiting coagulation factors .
every new anticoagulant therapy patient should be tested for inhibition at the commencement of therapy .
the accuracy and harmonization of patient inr results for different reagents is of the utmost importance for scientific publications , medication , and patient care .
today 's measuring principle is taking the sum of active coagulation factors ( fii , fvii , fx ) , and inhibition of inactive coagulation factors is not a satisfactory approach from the standpoint of accurate patient care .
the errors in inrs are too great , which in many ways affects the success of medication and patient well - being , also using the same calibration ( local calibration ) .
the new - generation pt offers possibilities of more accurate inr results and patient care based on control of active coagulation factors .
the therapeutic inr ranges guiding anticoagulant therapy using  old methods  are based on the principle that both calibrators and patient samples have inactive coagulation factors ( inhibition ) on average , which compensate for each other in the final inr result .
the therapeutic range lies between thrombosis and bleeding , and complications are serious and general .
the medication and care of oat patients must be rendered better and safe using a more sensitive pt test .
 warfarin is the most widely used medicine for oral anticoagulant therapy ( oat ) . 
 it inhibits the synthesis of coagulation factors ii , vii , ix , and x in the liver and results in the production of inactive or partially active versions of these factors . 
 inactive coagulation factors interfere with prothrombin time measurement ( quick and owren pt ) measuring the sum of coagulation activity and inhibition . 
 the narrow therapeutic range here involves a danger of serious complications and the risk of bleeding or thrombosis . 
 the new - generation pt method can measure coagulation activity and inhibition separately . 
 this new technique promotes patient care and anticoagulant medication ( warfarin , dicoumarol ) based on coagulation activity in vivo 
 . both therapy and laboratory controls should be unquestionably accurate and based solely on in vivo coagulation activity . 
 inactive coagulation factors ( inhibition ) render measurement , calibration , and harmonization . 
 the use of the new - generation pt method based on measurement of coagulation activity in vivo could develop vitamin k antagonist ( vka ) therapy for the marked benefit of patients . 
History
Warfarin
International Normalized Ratio
ISI calibration
The new-generation prothrombin time method
Conclusions