Heparin
238115
225641738
2008-07-14T18:15:58Z
TJRC
4033135
/* Heparin structure */ more detail; no need to give once-used reference a name
{{Drugbox|
| IUPAC_name = see [[#Heparin structure|''Heparin structure'']]
| image=Heparin-2D-skeletal.png
| image2=Heparin-3D-vdW.png
| CAS_number=9005-49-6
| ATC_prefix=B01
| ATC_suffix=AB01
| ATC_supplemental={{ATC|C05|BA03}} {{ATC|S01|XA14}}
| PubChem=772
| DrugBank=APRD00056
| C = 12 | H = 19 | N = 1 | O = 20 | S = 3
| molecular_weight = 12000–15000 g/mol
| bioavailability= nil
| metabolism = hepatic
| elimination_half-life=1.5 hrs
| excretion = ?
| pregnancy_category = C
| legal_status = ?
| routes_of_administration= i.v., s.c.
}}
'''Heparin''', a highly-sulfated [[glycosaminoglycan]], is widely used as an injectable [[anticoagulant]] and has the highest negative [[charge density]] of any known [[biomolecule|biological molecule]].<ref name= cox>{{cite book |last = Cox | first = M. | coauthors = Nelson D. | title = Lehninger, Principles of Biochemistry | issue=4 | publisher = Freeman | date = 2004 | pages=1100 | id=ISBN 0-71674339-6 }}</ref> It can also be used to form an inner anticoagulant surface on various experimental and medical devices such as [[test tube]]s and [[renal dialysis]] machines. Pharmaceutical grade heparin is derived from [[mucosal]] tissues of [[slaughterhouse|slaughtered]] meat animals such as [[porcine]] (pig) intestine or [[bovine]] (cow) lung.<ref>{{cite journal | author = Linhardt RJ, Gunay NS. | title = Production and Chemical Processing of Low Molecular Weight Heparins | journal = Sem. Thromb. Hem. | year=1999 | volume=3 | pages=5–16 | pmid = 10549711 }}</ref>
Although used principally in medicine for anticoagulation, the true physiological role in the body remains unclear, because blood anti-coagulation is achieved mostly by endothelial cell-derived [[heparan sulfate]] proteoglycans.<ref>{{cite journal | author=Marcum JA, McKenney JB. ''et al''. | title= Anticoagulantly active heparin-like molecules from mast cell-deficient mice | journal= Am. J. Physiol. | year=1986 | volume=250 | issue=5 Pt 2 | pages=H879–888 | pmid =3706560}}</ref> Heparin is usually stored within the secretory granules of [[mast cell]]s and released only into the [[vasculature]] at sites of tissue injury. It has been proposed that, rather than anticoagulation, the main purpose of heparin is in a defensive mechanism at sites of tissue injury against invading bacteria and other foreign materials.<ref>{{cite journal | author=Nader, HB ''et al''. | title=Heparan sulfates and heparins: similar compounds performing the same functions in vertebrates and invertebrates? | journal= Braz. J. Med. Biol. Res. | year=1999 | volume=32 | issue=5 | pages=529–538 | pmid = 10412563 | doi=10.1590/S0100-879X1999000500005}}</ref> In addition, it is preserved across a number of widely different species, including some invertebrates which lack a similar blood coagulation system.
==Heparin structure==
Native heparin is a polymer with a molecular weight ranging from 3 [[atomic mass unit|kDa]] to 40 kDa, although the average molecular weight of most commercial heparin preparations is in the range of 12 kDa to 15 kDa.
Heparin is a member of the [[glycosaminoglycan]] family of [[carbohydrates]] (which includes the closely-related molecule [[heparan sulfate]]) and consists of a variably-sulfated repeating [[disaccharide]] unit.<ref>{{cite web | last = Bentolila | first = A. ''et al''. | title = Synthesis and heparin-like biological activity of amino acid-based polymers | publisher = Wiley InterScience | url = http://www3.interscience.wiley.com/cgi-bin/abstract/75500237/ABSTRACT?CRETRY=1&SRETRY=0 | accessdate = 2008-03-10}}</ref>
The main disaccharide units that occur in heparin are shown below. The most common disaccharide unit is composed of a 2-O-sulfated [[iduronic acid]] and 6-O-sulfated, N-sulfated glucosamine, IdoA(2S)-GlcNS(6S). For example, this makes up 85% of heparins from beef lung and about 75% of those from porcine intestinal mucosa.<ref>{{cite journal | author= Gatti, G., Casu, B. ''et al''. | title = Studies on the Conformation of Heparin by <sup>l</sup>H and <sup>13</sup>C NMR Spectroscopy | journal= Macromolecules | year=1979 | volume=12 | issue=5 | pages=1001–1007 | url=http://pubs.acs.org/cgi-bin/abstract.cgi/mamobx/1979/12/i05/f-pdf/f_ma60071a044.pdf | doi = 10.1021/ma60071a044}}</ref>
Not shown below are the rare disaccharides containing a 3-O-sulfated glucosamine (GlcNS(3S,6S)) or a free amine group (GlcNH<sub>3</sub><sup>+</sup>). Under physiological conditions, the [[ester]] and [[amide]] sulfate groups are deprotonated and attract positively-charged counterions to form a heparin salt. It is in this form that heparin is usually administered as an anticoagulant.
1 unit of heparin (the "[[William Henry Howell|Howell]] Unit") is an amount approximately equivalent to 0.002 mg of pure heparin, which is the quantity required to keep 1 mL of cat's blood fluid for 24 hours at 0°C. <ref>{{Cite web|url=http://cancerweb.ncl.ac.uk/cgi-bin/omd?Howell+unit|title=Online Medical Dictionary|accessdate=2008-07-11|publisher=Centre for Cancer Education|year=2000}}</ref>
<center>
<gallery>
Image:GlcA-GlcNAc.png|<center>'''GlcA-GlcNAc'''</center>
Image:GlcA-GlcNS.png|<center>'''GlcA-GlcNS'''</center>
Image:IdoA-GlcNS.png|<center>'''IdoA-GlcNS'''</center>
Image:IdoA(2S)-GlcNS.png|<center>'''IdoA(2S)-GlcNS'''</center>
Image:IdoA-GlcNS(6S).png|<center>'''IdoA-GlcNS(6S)'''</center>
Image:IdoA(2S)-GlcNS(6S).png|<center>'''IdoA(2S)-GlcNS(6S)'''</center>
</gallery>
</center>
===Abbreviations===
*'''GlcA''' = β-D-[[glucuronic acid]]
*'''IdoA''' = α-L-[[iduronic acid]]
*'''IdoA(2S)''' = 2-O-sulfo-α-L-iduronic acid
*'''GlcNAc''' = 2-deoxy-2-acetamido-α-D-glucopyranosyl
*'''GlcNS''' = 2-deoxy-2-sulfamido-α-D-glucopyranosyl
*'''GlcNS(6S)''' = 2-deoxy-2-sulfamido-α-D-glucopyranosyl-6-O-sulfate
===Three-dimensional structure===
The three-dimensional structure of heparin is complicated by the fact that [[iduronic acid]] may be present in either of two low-energy conformations when internally positioned within an oligosaccharide. The conformational equilibrium being influenced by sulfation state of adjacent glucosamine sugars.<ref>{{cite journal | author=Ferro D, Provasoli A, ''et al''. | title=Conformer populations of L-iduronic acid residues in glycosaminoglycan sequences | journal = Carbohydr. Res. | year=1990 | volume=195 | pages=157–167 | pmid = 2331699 | doi=10.1016/0008-6215(90)84164-P}}</ref> Nevertheless, the solution structure of a heparin dodecasacchride composed solely of six GlcNS(6S)-IdoA(2S) repeat units has been determined using a combination of NMR spectroscopy and molecular modeling techniques.<ref>{{cite journal | author = Mulloy B, Forster MJ, Jones C, Davies DB. | title = NMR and molecular-modelling studies of the solution conformation of heparin | journal = Biochem. J. | year=1993 | volume=293 | pages=849–858 | url = http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=8352752 | pmid = 8352752 }}</ref> Two models were constructed, one in which all IdoA(2S) were in the <sup>2</sup>S<sub>0</sub> conformation ('''A''' and '''B''' below), and one in which they are in the <sup>1</sup>C<sub>4</sub> conformation ('''C''' and '''D''' below). However there is no evidence to suggest that changes between these conformations occur in a concerted fashion. These models correspond to the protein data bank code [http://www.rcsb.org/pdb/files/1hpn.pdb 1HPN.]
<center>[[Image:Heparin-3D-structures.png|600px|Two different structures of heparin]]</center>
In the image above:
*'''A''' = 1HPN (all IdoA(2S) residues in <sup>2</sup>S<sub>0</sub> conformation) [http://wiki.jmol.org:81/index.php/User:K.murphy Jmol viewer]
*'''B''' = [[van der Waals radius]] space filling model of ''A''
*'''C''' = 1HPN (all IdoA(2S) residues in <sup>1</sup>C<sub>4</sub> conformation) [http://wiki.jmol.org:81/index.php/User:K.murphy Jmol viewer]
*'''D''' = van der Waals radius space filling model of ''C''
In these models, heparin adopts a helical conformation, the rotation of which places clusters of sulfate groups at regular intervals of about 17 [[angstrom]]s (1.7 [[nanometer|nm]]) on either side of the helical axis.
==Medical use==
Heparin is a naturally-occurring anticoagulant produced by [[basophil]]s and [[mast cell]]s.<ref>{{cite book |last = Guyton | first = A. C. | coauthors = Hall, J. E. | title = Textbook of Medical Physiology | issue=11 | publisher = Elsevier Saunders | date = 2006 | pages=464 | id=ISBN 0-7216-0240-1}}</ref> Heparin acts as an anticoagulant, preventing the formation of clots and extension of existing clots within the blood. While heparin does not break down clots that have already formed (unlike [[tissue plasminogen activator]]), it allows the body's natural [[Fibrinolysis|clot lysis]] mechanisms to work normally to break down clots that have already formed.
Heparin is used for anticoagulation for the following conditions:
*[[Acute coronary syndrome]], e.g., [[myocardial infarction]]
*[[Atrial fibrillation]]
*[[Deep-vein thrombosis]] and [[pulmonary embolism]]
*[[Cardiopulmonary bypass]] for [[heart surgery]].
Heparin and its derivatives ([[enoxaparin]], [[dalteparin]], and so forth) are effective at preventing deep-vein thromboses and pulmonary emboli in patients at risk,<ref>{{cite journal|author=Agnelli G, Piovella F, Buoncristiani P, ''et al.''|title=Enoxaparin plus compression stockings compared with compression stockings alone in the prevention of venous thromboembolism after elective neurosurgery|journal=N Engl J Med|year=1998|volume=339|issue=2|pages=80–5|pmid=9654538|doi=10.1056/NEJM199807093390204}}</ref><ref>{{cite journal|volume=346|pages=975–980|year=2002|issue=13|title=Duration of prophylaxis against venous thromboembolism with enoxaparin after surgery for cancer|author=Bergqvist D, Agnelli G, Cohen AT, ''et al.''|url=http://content.nejm.org/cgi/content/abstract/346/13/975|journal=N Engl J Med|doi=10.1056/NEJMoa012385|pmid=11919306}}</ref> but there is no evidence that they are effective at preventing death.<ref>{{cite journal|author=Handoll HHG, Farrar MJ, McBirnie J, Tytherleigh-Strong G, Milne AA, Gillespie WJ|title=Heparin, low molecular weight heparin and physical methods for preventing deep vein thrombosis and pulmonary embolism following surgery for hip fractures|journal=Cochrane Database Syst Rev|year=2002|volume=4|pages=CD000305|doi=10.1002/14651858.CD000305}}</ref> Current guidelines recommend aspirin and leg stockings instead.<ref>{{cite web|author=Scottish Intercollegiate Guidelines Network|title=Prophylaxis of venous thromboembolism|location=Edinburgh|publisher=Scottish Intercollegiate Guidelines Network|year=2002|url=http://www.sign.ac.uk/guidelines/fulltext/62/index.html|accessdate=2008-06-08}}</ref>
===Mechanism of anticoagulant action===
Heparin binds to the enzyme inhibitor [[antithrombin]] (AT) causing a conformational change that results in its activation through an increase in the flexibility of its reactive site loop.<ref>{{cite journal | author = Chuang YJ, Swanson R. ''et al''. | title = Heparin enhances the specificity of antithrombin for thrombin and factor Xa independent of the reactive center loop sequence. Evidence for an exosite determinant of factor Xa specificity in heparin-activated antithrombin | journal= J. Biol. Chem. | year= 2001 | volume= 276 | issue= 18 | pages= 14961–14971 | pmid = 11278930 | doi = 10.1074/jbc.M011550200}}</ref> The activated AT then inactivates [[thrombin]] and other proteases involved in blood clotting, most notably [[factor Xa]]. The rate of inactivation of these proteases by AT can increase by up to 1000-fold due to the binding of heparin.<ref>{{cite journal | author = Bjork I, Lindahl U. | title = Mechanism of the anticoagulant action of heparin | url=http://www.springerlink.com/content/g67115564280w013/ | journal=Mol. Cell. Biochem. | year=1982 | volume=48 | pages=161–182 | doi = 10.1007/BF00421226}}</ref>
AT binds to a specific pentasaccharide sulfation sequence contained within the heparin polymer
GlcNAc/NS(6S)-GlcA-GlcNS(3S,6S)-IdoA(2S)-GlcNS(6S)
The conformational change in AT on heparin-binding mediates its inhibition of factor Xa. For thrombin inhibition however, thrombin must also bind to the heparin polymer at a site proximal to the pentasaccharide. The highly-negative charge density of heparin contributes to its very strong [[electrostatic]] interaction with [[thrombin]].<ref name=cox/> The formation of a [[ternary complex]] between AT, thrombin, and heparin results in the inactivation of thrombin. For this reason heparin's activity against thrombin is size-dependent, the ternary complex requiring at least 18 saccharide units for efficient formation.<ref>{{cite journal | author = Petitou M, Herault JP, Bernat A, Driguez PA, ''et al''. | title= Synthesis of Thrombin inhibiting Heparin mimetics without side effects | journal= Nature | year=1999 | volume=398 | pages=417–422 | pmid= 10201371 | doi= 10.1038/18877}}</ref> In contrast anti factor Xa activity only requires the pentasaccharide binding site.
[[Image:fondaparinux.jpeg|left|thumb|500px|Chemical structure of [[fondaparinux]]]]
This size difference has led to the development of [[low molecular weight heparin|low-molecular-weight heparin]]s (LMWHs) and more recently to [[fondaparinux]] as pharmaceutical anticoagulants. Low-molecular-weight heparins and fondaparinux target anti-factor Xa activity rather than anti-thrombin (IIa) activity, with the aim of facilitating a more subtle regulation of coagulation and an improved therapeutic index. The chemical structure of fondaparinux is shown to the left. It is a synthetic pentasaccharide, whose chemical structure is almost identical to the AT binding pentasaccharide sequence that can be found within polymeric heparin and [[heparan sulfate]].
With LMWH and fondaparinux, there is a reduced risk of [[osteoporosis]] and [[heparin-induced thrombocytopenia]] (HIT). Monitoring of the [[APTT]] is also not required and indeed does not reflect the anticoagulant effect, as APTT is insensitive to alterations in factor Xa.
[[Danaparoid]], a mixture of [[heparan sulfate]], [[dermatan sulfate]], and [[chondroitin sulfate]] can be used as an anticoagulant in patients who have developed HIT. Because danaparoid does not contain heparin or heparin fragments, cross-reactivity of danaparoid with heparin-induced antibodies is reported as less than 10%.<ref>Shalansky, Karen. [http://www.vhpharmsci.com/Newsletters/1990s-NEWS/article6.htm DANAPAROID (Orgaran®) for Heparin-Induced Thrombocytopenia.] Vancouver Hospital & Health Sciences Centre, February 1998 Drug & Therapeutics Newsletter. Retrieved on [[2007-01-08]].</ref>
The effects of heparin are measured in the lab by the partial thromboplastin time ([[aPTT]]), (the time it takes the [[blood plasma]] to clot).
===Administration===
Details of administration are available in [[clinical practice guidelines]] by the [[American College of Chest Physicians]]:<ref name="pmid15383472">{{cite journal |author=Hirsh J, Raschke R |title=Heparin and low-molecular-weight heparin: the Seventh ACCP Conference on Antithrombotic and Thrombolytic Therapy |journal=Chest |volume=126 |issue=3 Suppl |pages=188S–203S |year=2004 |pmid=15383472 |doi=10.1378/chest.126.3_suppl.188S}}</ref>
* [http://www.chestjournal.org/cgi/content/full/126/3_suppl/188S/T4 Non-weight-based heparin dose adjustment]
* [http://www.chestjournal.org/cgi/content/full/126/3_suppl/188S/T5 Weight-based-heparin dose adjustment]
Heparin is given [[parenteral]]ly, as it is degraded when taken by mouth. It can be injected intravenously or subcutaneously (under the skin). Intramuscular injections (into muscle) are avoided because of the potential for forming [[hematoma]]s.
Because of its short biologic [[half-life]] of approximately one hour, heparin must be given frequently or as a continuous [[infusion]]. However, the use of [[low molecular weight heparin]] (LMWH) has allowed once daily dosing, thus not requiring a continuous infusion of the drug. If long-term anticoagulation is required, heparin is often used only to commence anticoagulation therapy until the oral anticoagulant [[warfarin]] takes effect.
===Adverse reactions===
A serious side-effect of heparin is [[heparin-induced thrombocytopenia]] (HIT syndrome). HITS is caused by an immunological reaction that makes [[platelet]]s a target of immunological response, resulting in the degradation of platelets. This is what causes thrombocytopenia. This condition is usually reversed on discontinuation, and can generally be avoided with the use of synthetic heparins. There is also a benign form of thrombocytopenia associated with early heparin use, which resolves without stopping heparin.
Rarer side-effects include [[alopecia]] and [[osteoporosis]] with chronic use.
As with many drugs, overdoses of heparin can be fatal. In September 2006, heparin received worldwide publicity when 3 prematurely-born infants died after they were mistakenly given overdoses of heparin at an Indianapolis hospital.<ref>{{cite news | last = Kusmer | first = Ken | publisher = Fox News (Associated Press) | title = 3rd Ind. preemie infant dies of overdose | url= http://www.foxnews.com/story/0,2933,214729,00.html | date= [[2006-09-20]] | accessdate = 2007-01-08}}</ref>
===Treatment of overdose===
In case of overdose, [[protamine sulfate]] can be given to counteract the action of heparin.
==History==
Heparin is one of the oldest drugs currently still in widespread clinical use. Its discovery in 1916 predates the establishment of the [[Food and Drug Administration|United States Food and Drug Administration]], although it did not enter clinical trials until 1935.<ref>{{cite journal | author=Linhardt RJ. | title = Heparin: An important drug enters its seventh decade | journal = Chem. Indust. | year=1991 | volume=2 | pages=45–50}}</ref> It was originally isolated from canine [[liver]] cells, hence its name (''hepar'' or "ηπαρ" is Greek for "liver"). Heparin's discovery can be attributed to the research activities of two men, [[Jay McLean]] and [[William Henry Howell]].
In 1916, McLean, a second-year medical student at [[Johns Hopkins University]], was working under the guidance of Howell investigating pro-coagulant preparations, when he isolated a fat-soluble phosphatide anti-coagulant. It was Howell who coined the term ''heparin'' for this type of fat-soluble anticoagulant in 1918. In the early 1920s, Howell isolated a water-soluble polysaccharide anticoagulant, which was also termed ''heparin'', although it was distinct from the phosphatide preparations previously isolated. It is probable that the work of McLean changed the focus of the Howell group to look for anticoagulants, which eventually led to the polysaccharide discovery.
Between 1933 and 1936, Connaught Medical Research Laboratories, then a part of the [[University of Toronto]], perfected a technique for producing safe, non-toxic heparin that could be administered to patients in a salt solution. The first human trials of heparin began in May 1935, and, by 1937, it was clear that Connaught's heparin was a safe, easily-available, and effective blood anticoagulant. Prior to 1933, heparin was available, but in small amounts, and was extremely expensive, toxic, and, as a consequence, of no medical value.<ref>{{cite web | last = Rutty | first = CJ | title = Miracle Blood Lubricant: Connaught and the Story of Heparin, 1928–1937 | publisher = Health Heritage Research Services | url =http://www.healthheritageresearch.com/Heparin-Conntact9608.html | accessdate = 2007-05-21}}</ref>
For a full discussion of the events surrounding heparin's discovery see Marcum J. (2000).<ref>{{cite journal | author = Marcum J. | title = The origin of the dispute over the discovery of heparin | journal = J. Hist. Med. Allied. Sci. | year=2000 | volume=55 | part=1 | pages=37–66 | pmid = 10734720 | doi = 10.1093/jhmas/55.1.37}}</ref>
==Novel drug development opportunities for heparin==
As detailed in the table below, there is a great deal of potential for the development of heparin-like structures as [[drug]]s to treat a wide range of [[disease]]s, in addition to their current use as [[anticoagulant]]s.<ref name=lever>{{cite journal | author=Lever R. and Page C.P. | title = Novel drug opportunities for heparin | journal= Nat. Rev. Drug Discov. | year=2002 | volume=1 | issue=2 | pages=140–148 | pmid = 12120095 | doi=10.1038/nrd724}}</ref><ref name=coombe>{{cite journal | author=Coombe D.R and Kett W.C. | title = Heparan sulfate-protein interactions: therapeutic potential through structure-function insights | journal= Cell. Mol. Life Sci. | year=2005 | volume=62 | issue=4 | pages=410–424 | pmid = 15719168 | doi = 10.1007/s00018-004-4293-7}}</ref>
{| class="wikitable" style="text-align:left"
|-valign="top"
| |'''Disease states sensitive to heparin'''||'''Heparins effect in experimental models''' || '''Clinical status'''
|-
|-valign="top"
|| [[Adult respiratory distress syndrome]] || Reduces cell activation and accumulation in airways, neutralizes mediators and cytotoxic cell products, and improves lung function in animal models || Controlled [[clinical trial]]s
|-
|-valign="top"
|| [[encephalomyelitis|Allergic encephalomyelitis]] || Effective in [[animal model]]s|| -
|-
|-valign="top"
|| [[Allergic rhinitis]] || Effects as for adult respiratory distress syndrome, although no specific nasal model has been tested || Controlled clinical trial
|-
|-valign="top"
|| [[Arthritis]] || Inhibits cell accumulation, [[collagen]] destruction and [[angiogenesis]] || [[Anecdotal report]]
|-
|-valign="top"
|| [[Asthma]] || As for adult respiratory distress syndrome, however it has also been shown to improve lung function in experimental models || Controlled clinical trials
|-
|-valign="top"
|| [[Cancer]] || Inhibits [[tumour]] growth, [[metastasis]] and angiogenesis, and increases survival time in animal models || Several anecdotal reports
|-
|-valign="top"
|| [[Hypersensitivity#Type_4_-_cell-mediated_.28Delayed-Type_Hypersensitivity.2C_DTH.29|Delayed type hypersensitivity reactions]] || Effective in animal models || -
|-
|-valign="top"
|| [[Inflammatory bowel disease]] || Inhibits inflammatory cell transport in general. No specific model tested || Controlled clinical trials
|-
|-valign="top"
|| [[Interstitial cystitis]] || Effective in a human experimental model of interstitial cystitis || Related molecule now used clinically
|-
|-valign="top"
|| [[Transplant rejection]] || Prolongs [[allograph]] survival in animal models || -
|}
- indicates no information available
As a result of heparin's effect on such a wide variety of disease states a number of drugs are indeed in development whose molecular structures are identical or similar to those found within parts of the polymeric heparin chain.<ref name=lever/>
{| class="wikitable" style="text-align:left"
|-valign="top"
| |'''Drug molecule'''||'''Effect of new drug compared to heparin''' || '''Biological activities'''
|-
|-valign="top"
|| Heparin tetrasaccharide || Non-anticoagulant, non-immunogenic, orally active || Anti-allergic
|-
|-valign="top"
|| [[Pentosan polysulfate]] || Plant derived, little anticoagulant activity, Anti-inflammatory, orally active|| Anti-inflammatory, anti-adhesive, anti-metastatic
|-
|-valign="top"
|| Phosphomannopentanose sulfate || Potent [[inhibitor]] of [[heparanase]] activity || Anti-metastatic, anti-angiogenic, anti-inflammatory
|-
|-valign="top"
|| Selectively chemically O-desulphated heparin || Lacks anticoagulant activity || Anti-inflammatory, anti-allergic, anti-adhesive
|-
|}
==De-polymerisation techniques==
Either chemical or enzymatic de-polymerisation techniques or a combination of the two underlie the vast majority of analyses carried out on the structure and function of heparin and [[heparan sulfate]] (HS).
===Enzymatic===
The enzymes traditionally used to digest heparin or HS are naturally produced by the soil bacterium ''Pedobacter heparinus'' (formerly named ''Flavobacterium heparinum'').<ref>{{cite journal | author= Shaya D, Tocilj A. ''et al''. | title = Crystal structure of heparinase II from Pedobacter heparinus and its complex with a disaccharide product | journal= J. Biol. Chem. | year=2006 | volume=281 | issue=22 | pages=15525–15535 | pmid = 16565082 | doi = 10.1074/jbc.M512055200}}</ref> This bacterium is capable of utilizing either heparin or HS as its sole carbon and nitrogen source. In order to do this it produces a range of enzymes such as [[lyase]]s, [[glucuronidase]]s, [[sulfoesterase]]s and [[sulfamidase]]s.<ref>{{cite journal | author= Galliher PM, Cooney CL. ''et al''. | title = Heparinase production by Flavobacterium heparinum | journal= Appl. Environ. Microbiol. | year=1981 | volume=41 | issue=2 | pages=360–365 | pmid = 7235692}}</ref> It is the lyases that have mainly been used in heparin/HS studies. The bacterium produces three lyases, heparinases I ({{EC number|4.2.2.7}}), II (no [[EC number]] assigned) and III ({{EC number|4.2.2.8}}) and each has distinct substrate specificities as detailed below.<ref>{{cite journal | author= Linhardt RJ, Turnbull JE. ''et al''. | title = Examination of the substrate specificity of heparin and heparan sulfate lyases | journal= Biochemistry | year=1990 | volume=29 | issue=10 | pages=2611–2617 | pmid = 2334685 | doi = 10.1021/bi00462a026}}</ref><ref>{{cite journal | author= Desai UR, Wang HM. and Linhardt RJ. | title = Specificity studies on the heparin lyases from Flavobacterium heparinum | journal= Biochemistry | year=1993 | volume=32 | issue=32 | pages=8140–8145 | pmid = 8347612 | doi = 10.1021/bi00083a012}}</ref>
{| class="wikitable" style="text-align:left"
|-valign="top"
| |'''Heparinase enzyme'''||'''Substrate specificity'''
|-
|-valign="top"
|| [[Heparinase I]] || GlcNS(±6S)-IdoA(2S)
|-
|| Heparinase II || GlcNS/Ac(±6S)-IdoA(±2S)<br> GlcNS/Ac(±6S)-GlcA
|-
|| [[Heparinase III]] || GlcNS/Ac(±6S)-GlcA/IdoA (with a preference for GlcA)
|}
[[Image:UA(2S)-GlcNS(6S).png|thumb|300px|right|UA(2S)-GlcNS(6S)]]
The lyases cleave heparin/HS by a [[beta elimination]] mechanism. This action generates an unsaturated double bond between C4 and C5 of the uronate residue.<ref>{{cite journal | author= Linker A, Hovingh P.| title = Isolation and characterization of oligosaccharides obtained from heparin by the action of heparinase | journal= Biochemistry | year=1972 | volume=11 | issue=4 | pages=563–568 | pmid = 5062409| doi = 10.1021/bi00754a013}}</ref><ref>{{cite journal | author= Linhardt RJ, Rice KG. ''et al''. | title = Mapping and quantification of the major oligosaccharide components of heparin | journal= Biochem. J. | year=1988 | volume=254 | issue=3 | pages=781–787 | pmid = 3196292}}</ref> The C4-C5 unsaturated uronate is termed ΔUA or UA. It is a sensitive UV [[chromaphore]] (max absorption at 232nm) and allows the rate of an enzyme digest to be followed as well as providing a convenient method for detecting the fragments produced by enzyme digestion.
===Chemical===
[[Nitrous acid]] can be used to chemically de-polymerise heparin/HS. Nitrous acid can be used at pH 1.5 or at a higher pH of 4. Under both conditions nitrous acid effects deaminative cleavage of the chain.<ref>{{cite journal | author= Shively JE, Conrad HE. | title = Formation of anhydrosugars in the chemical depolymerization of heparin | journal= Biochemistry | year=1976 | volume=15 | issue=18 | pages=3932–3942 | pmid = 9127 | doi = 10.1021/bi00663a005}}</ref> [[Image:reduction fig.png|thumb|right|IdoA(2S)-aMan: The anhydromannose can be reduced to an anhydromannitol]] At both 'high' (4) and 'low' (1.5) pH, deaminative cleavage occurs between GlcNS-GlcA and GlcNS-IdoA, all be it at a slower rate at the higher pH. The deamination reaction, and therefore chain cleavage, is regardless of O-sulfation carried by either monosaccharide unit.
At low pH deaminative cleavage results in the release of inorganic SO<sub>4</sub>, and the conversion of GlcNS into [[anhydromannose]] (aMan). Low pH nitrous acid treatment is an excellent method to distinguish N-sulfated polysaccharides such as heparin and HS from non N-sulfated polysacchrides such as [[chondroitin sulfate]] and [[dermatan sulfate]]; chondroitin sulfate and dermatan sulfate being un-susceptable to nitrous acid cleavage.
==Evolutionary conservation==
In addition to the bovine and porcine tissue from which pharmaceutical-grade heparin is commonly extracted, heparin has also been extracted and characterised from the following species:
<div class="references" style="-moz-column-count:3; column-count:3;">
#[[turkey (bird)| Turkey]].<ref>{{cite journal | author= Warda M, Mao W. ''et al''. | title = Turkey intestine as a commercial source of heparin? Comparative structural studies of intestinal avian and mammalian glycosaminoglycans. | journal= Comp. Biochem. Physiol. B Biochem. Mol. Biol. | year=2003 | volume=134 | issue=1 | pages=189–197 | pmid = 12524047 | doi = 10.1016/S1096-4959(02)00250-6}}</ref>
#[[Whale]].<ref>{{cite journal | author= Ototani N, Kikuchi M, Yosizawa Z. | title = Comparative studies on the structures of highly-active and relatively-inactive forms of whale heparin | journal= J Biochem (Tokyo) | year=1981 | volume=90 | issue=1 | pages=241–246 | pmid = 7287679}}</ref>
#[[Dromedary camel]].<ref>{{cite journal | author= Warda M, Gouda EM. ''et al''. | title = Isolation and characterization of raw heparin from dromedary intestine: evaluation of a new source of pharmaceutical heparin | journal= Comp. Biochem. Physiol. C Toxicol. Pharmacol. | year=2003 | volume=136 | issue=4 | pages=357–365 | pmid = 15012907 | doi= 10.1016/j.cca.2003.10.009}}</ref>
#[[Mouse]].<ref>{{cite journal | author= Bland CE, Ginsburg H. ''et al'' | title = Mouse heparin proteoglycan. Synthesis by mast cell-fibroblast monolayers during lymphocyte-dependent mast cell proliferation. | journal= J. Biol. Chem. | year=1982 | volume=257 | issue=15 | pages=8661–8666 | pmid = 6807978}}</ref>
#[[Humans]].<ref>{{cite journal | author= Linhardt RJ, Ampofo SA. ''et al'' | title = Isolation and characterization of human heparin | journal= Biochemistry | year=1992 | volume=31 | issue=49 | pages=12441–12445 | pmid = 1463730 | doi= 10.1021/bi00164a020}}</ref>
#[[Lobster]].<ref>{{cite journal | author= Hovingh P, Linker A. | title = An unusual heparan sulfate isolated from lobsters (Homarus americanus) | journal= J. Biol. Chem. | year=1982 | volume=257 | issue=16 | pages=9840–9844 | pmid = 6213614}}</ref>
#[[mussel| Fresh water mussel]].<ref>{{cite journal | author= Hovingh P, Linker A. | title = Glycosaminoglycans in ''Anodonta californiensis'', a freshwater mussel | journal=Biol. Bull | year=1993 | volume=185 | issue=2 | pages=263–276 | url=http://www.biolbull.org/cgi/content/abstract/185/2/263 | doi= 10.2307/1542006}}</ref>
#[[Clam]].<ref>{{cite journal | author= Pejler G, Danielsson A. ''et al'' | title =Structure and antithrombin-binding properties of heparin isolated from the clams Anomalocardia brasiliana and Tivela mactroides | journal= J. Biol. Chem. | year=1987 | volume=262 | issue=24 | pages=11413–11421 | pmid = 3624220}}</ref>
#[[Shrimp]].<ref>{{cite journal | author=Dietrich CP, Paiva JF. ''et al'' | title = Structural features and anticoagulant activities of a novel natural low-molecular-weight heparin from the shrimp Penaeus brasiliensis | journal= Biochim. Biophys. Acta. | year=1999 | volume=1428 | issue=2–3 | pages=273–283 | pmid = 10434045}}</ref>
#[[Mangrove crab]].<ref name=medeiros>{{cite journal | author=Medeiros GF, Mendes, A. ''et al'' | title = Distribution of sulfated glycosaminoglycans in the animal kingdom: widespread occurrence of heparin-like compounds in invertebrates | journal= Biochim. Biophys. Acta. | year=2000 | volume=1475 | issue=3 | pages=287–294 | pmid = 10913828}}</ref>
#[[Sand dollar]].<ref name=medeiros/>
</div>
The biological activity of heparin within species 6–11 is unclear and further supports the idea that the main physiological role of heparin is not anticoagulation. These species do not possess any blood coagulation system similar to that present within the species listed 1–5. The above list also demonstrates how heparin has been highly [[conserved sequence| evolutionarily conserved]] with molecules of a similar structure being produced by a broad range of organisms belonging to many different [[phylum|phyla]].
==Other uses/information==
*Heparin gel (topical) may sometimes be used to treat sports injuries. It is known that the diprotonated form of [[histamine]] binds site specifically to heparin.<ref>{{cite journal | author= Chuang W, Christ MD, Peng J, Rabenstein DL. | title = An NMR and molecular modeling study of the site-specific binding of histamine by heparin, chemically-modified heparin, and heparin-derived oligosacchrides | journal= Biochemistry. | year=2000 | volume=39 | pages=3542–3555 | doi= 10.1021/bi9926025 | pmid = 10736153}}</ref> The release of histamine from [[mast cells]] at a site of tissue injury contributes to an inflammatory response. The rationale behind the use of such topical gels may be to block the activity of released histamine, and so help to reduce inflammation.
*Heparin gains the capacity to initiate [[angiogenesis]] when its copper salt is formed. Copper-free molecules are non-angiogenic.<ref>{{cite journal | author= Alessandri, G. Raju, K. and Gullino, PM. | title = Mobilization of capillary endothelium ''in-vitro'' induced by effectors of angiogenesis ''in-vivo'' | journal= Cancer. Res. | year=1983 | volume=43 | pages=1790–1797 | pmid = 6187439}}</ref><ref>{{cite journal | author= Raju, K. Alessandri, G. Ziche, M. and Gullino, PM. | title = Ceruloplasmin, copper ions, and angiogenesis | journal= J. Natl. Cancer. Inst. | year=1982 | volume=69 | pages=1183–1188 | pmid = 6182332}}</ref> In contrast heparin may inhibit [[angiogenesis]] when it is administered in the presence of [[corticosteroids]].<ref>{{cite journal | author= Folkman J. | title = Regulation of angiogenesis: a new function of heparin | journal= Biochem. Pharmacol. | year=1985 | volume=34 | pages=905–909 | pmid = 2580535 | doi= 10.1016/0006-2952(85)90588-X}}</ref> This anti-angiogenic effect is independent of heparins anticoagulant activity.<ref>{{cite journal | author= Folkman J. and Ingber DE. | title = Angiostatic steroids. Method of discovery and mechanism of action | journal= Ann. Surg. | year=1987 | volume=206 | issue=3 | pages=374–383 | pmid = 2443088 | doi = 10.1097/00000658-198709000-00016}}</ref>
*Test tubes, [[Vacutainer]]s, and [[capillary]] tubes that use the [[lithium]] salt of heparin (lithium heparin) as an anticoagulant are usually marked with green stickers and green tops. Heparin has the advantage over [[EDTA]] as an anticoagulant, as it does not affect levels of most [[ion]]s. However it has been shown that the levels of ionized calcium may be decreased if the concentration of heparin in the blood specimen is too high.<ref>{{cite journal | author= Higgins, C. | title = The use of heparin in preparing samples for blood-gas analysis | journal= Medical Laboratory Observer | year=October 2007 | url=http://www.mlo-online.com/articles/1007/1007cover_story.pdf}}</ref> Heparin can interfere with some [[immunoassay]]s, however. As lithium heparin is usually used, a person's lithium levels cannot be obtained from these tubes; for this purpose, royal-blue-topped Vacutainers containing [[sodium]] heparin are used.
*[[Heparin-coated blood oxygenator]]s are available for use in heart-lung machines. Among other things, these specialized oxygenators are thought to improve overall [[biocompatibility]] and host homeostasis by providing characteristics similar to native endothelium.
*The DNA binding sites on [[RNA polymerase]] can be occupied by heparin, preventing the polymerase binding to promoter DNA. This property is exploited in a range of molecular biological assays.
*Common diagnostic procedures require [[PCR]] amplification of a patient's DNA, which is easily extracted from white blood cells treated with heparin. This poses a potential problem, since heparin may be extracted along with the DNA, and it has been found to interfere with the PCR reaction at levels as low as 0.002 U in a 50 μL reaction mixture.<ref>{{cite journal | author=Yokota M, Tatsumi N, Nathalang O, Yamada T, Tsuda I. | title = Effects of Heparin on Polymerase Chain Reaction for Blood White Cells | journal = J. Clin. Lab. Anal. | year=1999 | volume=13 | pages=133–140 | pmid = 10323479 | doi=10.1002/(SICI)1098-2825(1999)13:3<133::AID-JCLA8>3.0.CO;2-0}}</ref>
*Immobilized heparin can be used as an affinity [[ligand]] in [[protein purification]]. In this capacity, it can be used in two ways. The first of which is to use heparin to select out specific [[coagulation]] factors or other types of heparin-binding proteins from a complex mixture of non-heparin-binding proteins. Specific proteins can then be selectively dissociated from heparin with the use of differing salt concentrations or by use of a salt gradient. The second use is to use heparin as a high-capacity cation exchanger. This use takes advantage of heparins high number of anionic sulfate groups. These groups will capture common cations such as Na<sup>+</sup> or Ca<sup>2+</sup> in solution.
*Heparin does not break up fibrin, it only prevents conversion of fibrinogen to fibrin. Only thrombolytics can break up a clot.
==Controversies==
===Contamination recalls===
In December 2007, the FDA recalled a shipment of heparin because of a growth of [[Serratia marcescens]] in several unopened syringes of this product. The bacteria Serratia marcescens can lead to life-threatening injuries and/or death.<ref>[http://www.fda.gov/oc/po/firmrecalls/am2pat12_07.html ''AM2 PAT, Inc. Issues Nationwide Recall of Pre-Filled Heparin Lock Flush Solution USP (5 mL in 12 mL Syringes)''], Am2pat, Inc. Press release, [[December 20]], [[2007]]</ref>
In March 2008, major [[2008 Chinese export recalls|recalls]] of heparin were announced by the [[FDA]] due to contamination of the raw heparin stock imported from China.<ref>CBS News, [http://www.cbsnews.com/stories/2008/03/01/eveningnews/main3896578.shtml Blood-thinning drug under suspicion]</ref><ref>[http://www.fda.gov/CDER/drug/infopage/heparin/default.htm FDA informational page] with information and links about FDA investigation.</ref> The FDA admitted that it had violated its own policies by failing to inspect the American pharmaceutical firm [[Scientific Protein]]’s plant in China before approving the drug for sale.<ref name=NYT20080306>{{cite news |first=Gardiner | last=Harris | authorlink=http://topics.nytimes.com/top/reference/timestopics/people/h/gardiner_harris/index.html?inline=nyt-per | coauthors=Walt Bogdanich |title=Drug Tied to China Had Contaminant, F.D.A. Says | url=http://www.nytimes.com/2008/03/06/health/06heparin.html | publisher=[[New York Times]] | date=[[March 6]], [[2008]] | accessdate=2008-03-19}}</ref><ref>{{cite news | first=Gardiner | last=Harris | coauthors=Walt Bogdanich | title=German Authorities Report Problems With Blood Thinner | url= http://www.nytimes.com/2008/03/07/health/policy/07heparin.html | publisher=[[New York Times]] | date=[[March 7]], [[2008]] | accessdate=2008-06-11}}</ref> The U.S. Food and Drug Administration was quoted as stating that at least 81 deaths were believed linked to a raw heparin ingredient imported from the People's Republic of China, and that they had also received 785 reports of serious injuries associated with the drug’s use.<ref>{{cite news | first=Gardiner | last=Harris | title=U.S. Identifies Tainted Heparin in 11 Countries | url=http://www.nytimes.com/2008/04/22/health/policy/22fda.html | publisher=[[New York Times]] | date=[[April 22]], [[2008]] |accessdate=2008-06-11}}</ref> According to the New York Times, "Problems with heparin reported to the agency include difficulty breathing, nausea, vomiting, excessive sweating and rapidly falling blood pressure that in some cases led to life-threatening shock."<ref name=NYT20080306/>
The contaminant has been identified as an "over-sulphated" derivative of [[chondroitin sulfate]], a popular shellfish derived supplement often used for [[arthritis]].<ref>{{cite web|url=http://www.fda.gov/bbs/transcripts/2008/heparin_transcript_031908.pdf|title=FDA Media Briefing on Heparin|publisher=[[FDA]]|first=Julie|last=Zawisza|date=2008-03-29|accessdate=2008-04-23}}</ref> Since this "over-sulphated" variant is not naturally occurring and mimics the properties of heparin,the counterfeit is almost certainly intentional as opposed to an accidental lapse in manufacturing.<ref>{{cite news | first=Walt | last=Bogdanich | authorlink=http://topics.nytimes.com/top/reference/timestopics/people/b/walt_bogdanich/index.html?inline=nyt-per | title=Heparin Find May Point to Chinese Counterfeiting | url=http://www.nytimes.com/2008/03/20/health/20heparin.html?hp=&adxnnl=1&adxnnlx=1206032448-8vAmKaxtXzpFTH3rUaNgBg | publisher=[[The New York Times]] | date=[[March 20]], [[2008]] | accessdate=2008-03-20}}</ref> The heparin was cut from anywhere from 2-60% with a counterfeit substance due to cost effectiveness, and a shortage of suitable pigs in China.
When the FDA finally did conduct an inspection of Baxter's Chinese Heparin supplier, it found serious deficiencies at the facility which the FDA detailed in a letter to the Chinese company. The FDA has stated that it does not have the funds nor bear the responsibility to inspect on a regular basis overseas manufacturers of active pharmaceutical ingredients such as heparin.
===Use in homicide===
In 2006, [[Petr Zelenka (serial killer)|Petr Zelenka]], a nurse in the [[Czech Republic]], deliberately administered large doses to patients, killing 7, and attempting to kill 10 others.<ref>[http://www.radio.cz/en/article/85964 Nurse committed murders to "test" doctors], Radio Praha, [[May 12]], [[2006]]</ref>
===Overdose issues===
Actor [[Dennis Quaid]]'s twelve-day-old twins mistakenly were given an adult dosage, which is 1,000 times the recommended dosage for infants, in November 2007.<ref>Ornstein, Charles; Gorman, Anna. (November 21, 2007) [[Los Angeles Times]] ''[http://www.cnn.com/2007/SHOWBIZ/Movies/11/21/quaid.newborns.ap/index.html Report: Dennis Quaid's twins get accidental overdose.]''</ref> The overdose allegedly arose because the labeling and design of the adult and infant versions of the product were easily confused. The Quaid family subsequently sued the manufacturer, [[Baxter Healthcare Corp.]], for over $50,000.<ref>[http://www.usatoday.com/life/people/2007-12-04-quaid-lawsuit_N.htm Dennis Quaid and wife sue drug maker], USA Today, [[December 4]], [[2007]]</ref><ref>[http://www.latimes.com/features/health/la-me-quaid5dec05,1,1883436.story Dennis Quaid files suit over drug mishap], Los Angeles Times, [[December 5]], [[2007]]</ref>
In July 2008, another set of twins born at Christus Spohn Hospital South, a Texas hospital, died after an accidentally administered overdose of the drug. The overdose was due to a mixing error at the hospital pharmacy and, unlike the Quaid case, was unrelated to the product's packaging or labeling.<ref>[http://christusspohn.netreturns.biz/NewsReleases/Article_Detail.aspx?id=b8250e15-c437-48f3-8345-edc85a47565a Statement by Dr. Richard Davis, Chief Medical Officer, CHRISTUS Spohn Health System]], [[July 10]], [[2008]]</ref> [[As of July 2008]], whether the deaths were due to the overdose is under investigation.<ref>[http://www.dallasnews.com/sharedcontent/dws/news/texassouthwest/stories/DN-bloodthinner_11tex.ART.State.Edition1.4d4aa44.html At a Glance Heparin Overdose at Hospital], Dallas Morning News, [[July 11]]. [[2008]]</ref><ref>[http://abcnews.go.com/GMA/Parenting/story?id=5346509&page=1 Officials Investigate Infants' Heparin OD at Texas Hospital}, ABC News, [[July 11]], [[2008]]</ref>
==Popular culture==
* Heparin was featured in [[Dan Brown]]'s novel ''[[Angels and Demons]]'', in which the intentional overdose of the drug was used in the murder of a significant character that was disguised to resemble a [[death]] by [[stroke]].
* Heparin was also featured in the television film [[The Andromeda Strain (2008 miniseries)|The Andromeda Strain]] as a way to stop a mysterious alien disease from clotting people's blood.
==References==
{{reflist|2}}
==External links==
* [http://www.healthheritageresearch.com/Heparin-Conntact9608.html History of heparin]
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