Heparin

1
Heparin

• Heparin is the most widely used drug in treating
  thromboembolic diseases

2
Heparin
3
Heparin - the molecule

• Heparin is a heterogeneous mixture of unbranced
  polysaccharide chains
• Alternating monosaccharide units of L-iduronic
  acid and D-glucosamine
• The molecule size in the natural extract is 2 to
  40 KDa
• One third of the polysaccharide chains contain a
  specific antithrombin binding pentasaccharide
  sequence

4
Heparin - the structure
5
Heparin - the source

• Preparations from porcine or bovine intestinal
  mucus
• Preparation from bovine lung

6
Heparin - biological function?

• Heparin can not be found in circulating blood
• Heparan sulphate, a heparin-like molecule is
  found on the endothelial cells covering the blood
  vessels
• Heparan sulphate has anticoagulant activity

7
Heparinmechanism of action

• Heparin exerts parts of its anticoagulant
  activity through interaction with antithrombin
• Antithrombin binds specifically to a
  pentsaccharide in heparin
• Binding to heparin induces a conformational
  change in the antithrombin, which accelerate the
  enzyme inhibition

8
Thrombin inhibition catalysed by heparin
P
AT
H
P
AT
R
H
R
IIa
IIa
P
P
AT
H
AT
H
R
R
IIa
IIa
9
FXa inhibition catalysed by heparin
P
AT
H
P
AT
R
H
R
Xa
Xa
P
P
AT
H
AT
H
R
R
Xa
Xa
10
Heparinunspecific interactions

• Heparin interact unspecific with -Platelet
  Factor 4 -Plasma proteins (alpha-1-acid glyc
  oprotein, histidine rich glycoprotein
  etc) - Vascular endothelial cells

11
HeparinUnspecific interactions

• Unspecific interactions limits heparins
  catalytic effect on antithrombin
• Unspecific interactions increase with increased
  molecular weight of heparin

12
Two forms of heparin

• Unfractionated (UF) Heparin
• Low Molecular Weight (LMW) Heparin

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UF heparin versus LMW Heparin
LMW Heparin
UF Heparin

• Average molecular weight 15 KDa
• 15-100 monosaccharides per molecule
• About 30 of the molecules contains the
  antithrombin binding pentasaccharide

• Average molecular weight between 4-6.5 KDa
• 4-40 monosaccharides per molecule
• Prepared from UF heparin by chemical or enzymatic
  depolymerization

14
UF heparin versus LMW Heparin
UF Heparin
LMW Heparin

• anti-FXaanti-FIIa ratio is 11
• High affinity for plasma proteins like PF4
• 30 bio-availability after subcutaneous injection

• anti-FXaanti-FIIa ratio is between 12 and 14
• Low affinity for plasma proteins like PF4
• 90 bio-availability after subcutaneous injection

15
UF heparin versus LMW Heparin

• Elimination primarily through cellular uptake
• Half-life is dose dependent (1-3 hours)
• Can be measured by APTT assays

• Elimination mainly through renal filtration
• Half-life around 4 hours
• Can not be measured by APTT assays

16
Trade names for LMW Heparin

• Fraxiparin/Seleparin (Sanfoni, France)
• Enoxaparin, Clexane, Lovenox (Rhone-Poulenc,
  France)
• Fragmin, Dalteparin (PharmaciaUpjohn, Sweden)
• Sandoparin, Certoparin (Sandoz, Germany)
• Logoparin, Innohep (Novo, Denmark)
• Innohep (Leo, Denmark)
• Reviparin, Clivarin (Knoll, Germany)
• Ardeparin, Normiflo (Wyeth, US)
• Merckle LMWH (Merckle, Germany)
• Fluxum (Oporcin, Italy)
• Boxol (Rovi, Spain)
• Miniparin (Syntex, Argentina)

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Differences between LMW heparin

• Average molecular weight
• Molecular weight distribution
• Content of glycoaminoglycans
• USP activity (U/mg)
• Anti-Xa activity (U/mg)
• Antithrombotic effects
• Bio-availability
• TFPI release

18
Heparinmode of administration

• Intravenous (immediate effect)
• Subcutaneous (effect within 20-60 minutes)

19
Heparin - clinical use

• Prophylactics of DVT and PE -Prevention of
  formation of thrombin -Low dose
  regimens -High risk groups (acute myocardial
  infarction, surgery)
• Treatment of DVT and PE -Prevention of further
  thrombin generation -High dose regimens

20
Heparin - clinical use

• Coronary heart disease -Unstable
  angina -Acute myocardial infarction -After
  thrombolytic therapy with rt-PA
• Artificial thrombogenic surfaces

21
UF Heparinclinical strategy in thrombosis

• Start with a bolus injection of 5000-1000 U
• Maintenance infusion at 1000-2000 U/h
• Check APTT at 6 h to keep APTT between 1.5 and
  2.5 times control (0.2-0.4 U/ml anti-FXa)
• Check platelet count daily
• Start warfarin therapy immediately
• Stop heparin therapy after 4-7 days
• Continue with warfarin for 3 months

22
Heparin - complications

• Haemorrhage
• Thrombocytopenia
• Osteoporosis (long term use)

23
UF heparin versus LMW Heparin - clinical benefits

• Fewer injections - LMWH is administered once
  daily
• Lower dose of LMWH
• LMWH gives more predictable response
• Less need for monitoring of LMWH
• Equal or better antithrombotic effects with LMWH
• Fewer bleeding complications?

24
Heparin - purpose oflaboratory monitoring

• Minimise the risk of haemorrhage
  caused by over-dosage
• Optimise the antithrombotic effect by appropriate
  dose adjustment

25
Heparin - individual variation in antithrombotic
response

• Age
• Weight
• Sex
• Drug interactions
• Associated disease
• Extent of fibrin
• Extent of heparin binding proteins
• Renal insufficiency (LMWH)

26
Heparin - clinical monitoring
27
Heparinwhen to monitor?

• High doses of intravenous UF heparin should be
  monitored continuously
• LMW Heparin is normally monitored in the
  beginning of the treatment
• Monitoring of heparin given as prophylactics in
  low dosage is only of informative value

28
Heparin measurements
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Heparin assays - APTT

• Principle -The sample is added to a
  mixture of phospholipid and surface
  activator -The reaction is triggered by
  addition of Ca2 -The clotting time is
  measured
• Treatment of DVT/PE -APTT ration of 1.5-2.5
• Prophylactics - high risk surgery -APTT ratio
  1-1.2

30
Heparin assays - APTT

• The most common test for UF heparin
• Does not work for LMW heparin
• Different APTT reagents gives different
  therapeutic range

31
Heparin assaysAPTT should not be used!

• During combined heparin-Warfarin therapy
• During combined heparin and rt-PA therapy
• In patients with lupus anticoagulant
• In patients with elevated FVIII
• In patients with elevated PF4
• To monitor LMW heparin

32
Heparin - principal for chromogenic assays
FXa or FIIa is added in excess to a mixture of
plasma and a chromogenic substrate. The assay
could be run either with or without addition of
exogenous AT. The enzyme could be added to the
reaction either before or after the substrate
AT Heparin
ATHeparin
ATHeparin FXa(excess)
AT-FXa Heparin FXa(residual)
FXa(residual)
Chromogenic substrate
Peptide pNA
33
Two-stage heparin assaysmeasures the heparin
concentration

• Addition of exogenous antithrombin
• Plasma and FXa is incubated before addition of
  the substrate
• Sensitive to the incubation time between plasma
  and FXa
• Plasma is always diluted

34
One-stage heparin assaysmeasures the heparin
activity

• No exogenous antithrombin
• Substrate is added before the FXa
• Insensitive to the first incubation time
• Often undiluted plasma

35
One-stage assays and two-stages assays give in
most cases the same results for ex vivo
samplesbut not for in vitro samples
36
Correlation CT Heparin versus CM Heparin
37
CorrelationCT LMW Heparin/Heparin versus CM
Heparin
38
CorrelationRotachrom LMW Heparin versus CM
Heparin
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CorrelationRotachrom Heparin versus CM Heparin
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CorrelationBerichrom Heparin versus CM Heparin
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CorrelationIL test Heparin versus CM Heparin
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In vitro effect of antithrombin activity for CM
Heparin
Antithrombin activity ()
Difference in measured heparin activity (IU/ml)
43
CorrelationCM Heparin with AT versus CM Heparin
without AT
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Difference in results CM Heparin /- AT ex vivo
Antithrombin activity ()
Difference in measured heparin activity (IU/ml)
45
Chromogenixs Heparin kits

• Coamatic Heparin
• Coatest Heparin
• Coatest LMW/LMW Heparin
• Coacute Heparin

46
Coatest Heparinkit configuration

• 1 vial of FXa, 71 nkat
• 1 vial of S-2222, 15 mg
• 1 vial of human antithrombin, 10 IU
• 1 vial of concentrated buffer, 10 ml
• 4 vials of normal human plasma, 1 ml/vial

47
Coatest LMW Heparin/Heparinkit configuration

• 1 vial of FXa, 13 nkat
• 1 vial of S-2732, 6 mg
• 1 vial of buffer, 20 ml
• 1 vial of LMW Heparin standard, 100 IU/ml

48
Coamatic Heparinkit configuration

• 2 vials of FXa, 35 nkat/vial
• 2 vials of S-2732, 15 mg/vial

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Coacute Heparinkit configuration

• 10 single test cuvettes, with FXa, S-2732 and
  antithrombin
• 2 empty cuvettes for blanking
• 10 tubes with buffer
• 1 vial of stop solution

50
Coatest Heparin

• Two-stage assay
• Addition of exogenous antithrombin corresponding
  to one plasma unit
• Sensitive to the first incubation time and
  thereby not applicable on all the new coagulation
  analysers
• Diluted assay
• Measuring range 0.05-0.7 IU/ml

51
Coatest LMW Heparin/Heparin

• One-stage assay
• No addition of antithrombin
• Insensitive to the first incubation time
• Undiluted plasma
• Non-linear standard curve
• Long reading time, thereby not applicable to all
  analysers
• Measuring range 0.05-1.0 IU/ml (log scale)

52
Coacute Heparin

• One-stage assay
• With additional antithrombin
• All reagents are co-lyophilised
• Stat analysis or for small hospitals
• Pre-calibrated curves included in the kit insert

53
Coamatic Heparin

• One-stage assay
• With or without additional antithrombin
  (industrial use?)
• Insensitive to the first incubation time
• Undiluted assay
• Applicable on almost all analysers
• Only three point calibration curve
• Binding to PF4 has been minimised

54
Coamatic Heparin

• Calibrators and controls, useful for all LMW
  heparin are available
• Superior stability, useful in both small and
  larger laboratories
• Resolution up to 2.0 IU/ml on most analysers
  (linear up to 0.8 IU/ml)
• Similar standard curves with different kinds of
  heparin

55
Coamatic HeparinSimilar standard curves with
most heparin
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Heparin assayscalibration

• The activity of UF heparin is expressed relative
  to the 4th or 5th international standard
• The activity of LMW heparin is expressed relative
  to the 1st international standard
• Coatest LMW Heparin/Heparin contains a LMW
  heparin standard calibrated against the 1st
  international standard

57
Heparin assayscalibration

• Normally recommended to use the same heparin for
  calibration as is administered to the patient
• Coamatic Heparin gives similar standard curves
  with all kinds of LMW Heparin, therefore it is
  possible to use our LMW Heparin calibrators and
  controls for all kinds of LMW Heparin

58
Coamatic Heparinkey sales arguments

• Independent of the first incubation time -
  suitable on all new coagulation analysers
• Undiluted assay- saves cuvettes and time
• Interference with PF4 is minimised by addition of
  dextran sulphate - sample handling is less
  critical

59
Coamatic Heparinkey sales arguments

• Only two components - easy and fast performance
• Excellent stability - useful in both the small
  and the large laboratory
• Similar standard curves with all kinds of heparin
  - similar resolution and precision no matter
  which heparin is tested

60
Coamatic Heparinkey sales arguments

• Excellent correlation with or without exogenous
  AT - the kit could be used independent of opinion
• Adapted to most analysers on the market - the
  assay could in most cases also be used on the
  customer backup instrument
• Calibrators and controls available