Department of Anaesthesia and Intensive Care Medicine, Royal Brompton and Harefield NHS Trust, Harefield Hospital, Harefield, Middlesex UB9 6JH, UK *Corresponding author
Abstract
Br J Anaesth 2002; 88: 84863
Keywords: blood, anticoagulants; blood, coagulation
This review concentrates on discussing the various therapeutic agents available to prevent or inhibit clot formation. Particular emphasis is placed on therapies associated with modification to coagulation factors, and the inhibitors of thrombin formation and action. The genesis of ischaemic cardiovascular disease is related to inappropriate platelet function and/or thrombin generation in excess amounts or at inappropriate sites. The most commonly used agents currently available to inhibit or slow thrombin production include vitamin K antagonists and heparin, acting through circulating or endothelial-derived intermediaries. More recently, a number of agents, which can act to directly inhibit thrombin, have been licensed for use in humans. It is expected that the range of these compounds will eventually grow to replace the use of unfractionated heparin (UFH) and vitamin K antagonists within the next few years. To better understand the mechanism of action of all of these compounds, a brief description of the mechanism of clot formation and the pivotal role of thrombin in this process is required together with the mechanisms for localizing and controlling this activity.
The coagulation cascade
The aim of the coagulation phase of haemostasis is the generation of fibrin strands that will bind and stabilize the weak platelet haemostatic plug. There are no covalent bonds holding the platelets together during the formation of the primary haemostatic plug. If left in this state the platelet plug, formed by platelet aggregation, would come apart in a few hours, resulting in late bleeding. The process of blood coagulation, with soluble factors in the blood entering into a chain of reactions that lead to the formation of fibrin, is intended to be localized to the area where the original platelet plug was formed.
This localization is achieved by two methods. First, the chain of reactions which led to the conversion of fibrinogen to fibrin are programmed to occur, and are most efficient and explosive, when restricted to a surface, such as platelet phospholipid. Second, there are a series of inhibitors that are intended to constrain the reaction to the site of injury and platelet deposition. These inhibition processes include the following.
1. Circulating factors such as antithrombin III (ATIII) and heparin cofactor II (HCII).
2. Those derived from endothelium such as tissue factor pathway inhibitor (TFPI).
3. The thrombomodulin system, which converts prothrombotic thrombin to an anticoagulant through the activation of circulating protein C.
Localization of the coagulation process
Historically, the blood coagulation system is divided into two initiating pathways: the tissue factor (extrinsic) pathway and the contact factor (intrinsic) pathway which meet at a final, common pathway, whereby factor Xa converts prothrombin to thrombin which then acts on fibrinogen. These pathways were identified and categorized during experiments to examine the effects of sufficiency and deficiency of the various circulating factors on assays of plasma coagulation. At present the immediate clinical investigation of haemostatic disorders still requires this compartmentalized, cascade-type model as the laboratory-based tests of coagulation focus on each of these separate aspects. The prothrombin time (PT) is a plasma and test-tube variant of the extrinsic pathway, and the activated clotting time (ACT) or activated partial thromboplastin time (aPTT) of the intrinsic system for blood and plasma, respectively.
This model based on the concept of a waterfall or cascade is an over simplification of the system, as proteins from each pathway can influence one another. It is probably more correct to think of the coagulation system as an interactive network with carefully placed amplifiers and restraints.
Fibrin formation is a process of initiation and amplification. The specific properties of platelets and the coagulation system cooperate to ensure that fibrin formation occurs only at the localized site where it is required to initiate wound repair. This is achieved by a number of physico-chemical means.
The surface of resting platelets contains acidic phospholipids such as phosphatidylserine that have their negatively charged pole directed inward. Spontaneous reversal of this charge is countered by a specific enzyme system in the platelet (a flipase), implying that this charge reversal is of pivotal importance. When the platelet becomes activated, the negatively charged phospholipid remains on the outside surface of the platelet membrane and is not flipped internally.
The coagulation system relies primarily on a group of soluble factors that circulate in the plasma. These factors are synthesized in the liver and expressed into the circulation (Table 1). Most coagulation factors are identified by Roman numerals, the active form denoted by the lower case a. They circulate in an inactive, zymogen form and become active after proteolytic cleavage. The exception to this is factor VII that can circulate as an active protease. Apart from factor XIII, which is a transglutaminase, all the active factors are serine protease related to the digestive enzyme, trypsin. Other factors in the coagulation process, such as tissue factor, factor V, factor VIII, and high molecular weight kininogen (HK) act as co-factors.
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Coagulation organization: amplification for explosive thrombin production
A recurrent theme in the coagulation system is the formation of activation complexes involving a serine protease, a zymogen or substrate, a co-factor, and an organizing surface, usually provided by the platelet membrane. These factors must be presented to each other in a tightly controlled way to ensure the process of coagulation is amplified and progressed with sufficient rapidity. This elegant organization can be appreciated by considering the formation of thrombin from prothrombin, which is the best characterized of such reactions.
It is axiomatic that production of thrombin needs to be explosive at the site of the injury in order to prevent it being washed away to cause havoc elsewhere in the vascular tree. The major problem to overcome in this process is to enable factor Xa to remove the restraint or protective bubble over the active site on prothrombin at a sufficient rate. Acceleration of the process is achieved by maximizing the encounter of active site and substrate by appropriate orientation of each molecule (Fig. 2A).
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The pivotal importance of these co-factors (V and VIII) becomes obvious when the kinetics of the reactions were considered. If we assume first that the rate constant for the conversion of prothrombin to thrombin by factor Xa in an aqueous solution is unity, then addition of calcium ions to this mixture will make the reaction rate 2.3 times faster. Activation of prothrombin by factor Xa is 22 times more likely to occur with both factor Xa and prothrombin combining on the negatively charged surface of platelet phospholipid than if the enzyme and substrate were just floating in solution. The addition of factor V to this mixture, by holding factor Xa and prothrombin in place and aligning them correctly, so they have no choice but to have a productive interaction, accelerates the reaction by 278 000-fold.
If similar kinetics are assumed for the activation of factor Xa, then simple mathematics show a colossal 7.7 million-fold acceleration of thrombin generation in a fully active system. Put another way, a fluid phase two-step coagulation process for the generation of thrombin, involving first the activation of factor X to Xa followed by the activation of prothrombin to thrombin would require about 3 months (89 days). This same reaction would take only 1 s in the unrestricted fully intact system. If the process works properly then explosive thrombin formation should occur only where it is directed and required.
Tissue factor and thrombin generation
Factor Xa can also be generated by a different, surface-dependent, pathway. The so-called tissue factor or extrinsic pathway is considered to be the principal initiating pathway of coagulation in vivo. In this reaction, factor X is cleaved by the serine protease, factor VII. Factor VII is held in the appropriate configuration by endothelial surface-bound tissue factor (Fig. 4).
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Circulating factor VII is an unusual coagulation protein for two reasons: (1) the non-activated (zymogen) form has some proteolytic activity and (2) about 1% of the circulating form exists as the active enzyme, factor VIIa. Either form can bind readily to TF and the complex thus formed will have enough activity to cleave factor X to factor Xa. Factor Xa will then rapidly convert the factor VII:TF complex to factor VIIa:TF and can thus potentiate the reaction. In addition to activating factor X, the factor VIIa:TF complex can also cleave factor IX to form factor IXa, which can then itself activate factor X as described above. This illustrates the lack of division of the contact and TF pathways in vivo.
Peripheral blood cells do not normally express TF. However, circulating monocytes and endothelial cells can be induced to produce TF by a variety of stimuli including endotoxin, tumour necrosis factor, interleukin 1, immune complexes, hypoxia, and hypothermia. This stimulation and expression of TF by phlogistic agents is thought to play a part in the consumptive coagulopathy associated with sepsis.
Thrombin and its activity
Thrombin is a serine protease that has become the focus of considerable research interest. This interest is driven by observations of the ubiquitous actions of thrombin and also by advances in our understanding of the molecular mechanisms involved in the structure and activity of this protease. In turn, this has lead to the development of agents that have either a direct effect on the cleavage site of the molecule or can in some other way inhibit the ability of thrombin to catalyse the conversion of fibrinogen to fibrin.
The thrombin molecule can be simplistically viewed as a sphere with a shaped groove along its equatorial axis (Fig. 5). The horizontal part of the groove extends around the molecule. The left-hand part of the horizontal section is included with the vertical area to make up the active proteolytic site. The extended horizontal groove, distal to the active site, is one of a number of anion binding exosites on the surface of the thrombin molecule. This specific exosite of thrombin is important as it is involved with thrombin inhibition/binding by the heparin: ATIII complex and also the carboxyl tail of hirudin (see below).
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Inhibitors of coagulation in humans
The process of thrombin generation must be localized and contained to prevent global thrombosis after minor injury. It is a basic tenet in biochemistry that every activator will have a cognate inhibitor and this is true for the coagulation system. The natural inhibitors fall into two main groups, endothelial or hepatic, based on their synthetic site. An alternate classification could be to separate the inhibitors into those that aim to inhibit thrombin production and those that directly inhibit this enzyme.
Endothelial-derived inhibitors
Endothelial-derived factors include TFPI and activated protein C (aPC). This latter protease has been investigated as a means of preventing thrombus formation or extension. The fascination with aPC and its generation are 2-fold. First, thrombin is responsible for the generation of aPC. Thrombin is held on the endothelial surface by a co-factor/receptor called thrombomodulin. The active site of the thrombin cleaves the protein C moiety to release aPC (Fig. 6). This clever device allows thrombin to be converted from a procoagulant to an anticoagulant protein. aPC is a serine protease that cleaves a peptide from the arms of factor V and factor VIII, thereby preventing appropriate participation in the tenase and prothrombinase complex (Fig. 7). Resistance to this cleavage is observed in patients who have a single point mutation in their factor V (so-called factor V Leiden). Similar to a genetic absence of protein C, this is not a lethal gene. However, patients with the Leiden mutation are at substantially increased risk of venous thrombosis,4 and myocardial infarction in certain populations.66 The second interest in the relationship between thrombin and protein C lies in the evolution of these proteins. Genomic mapping suggests that these proteins developed and evolved together. We also increasingly recognize a functional relationship between these proteins. One example is that cleavage by thrombin, to release the tethered ligand of the thrombin receptor, occurs at a site with structural and amino acid sequence homology with protein C.
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Individuals with congenital TFPI deficiency have not been identified. Mice homozygous for deletion of K1 in the TFPI gene die in utero implying that such a deficiency is not compatible with life. Of interest is that the inhibitor site of the K1 domain differs by only 1 amino acid residue from the inhibitor Kunitz domain of aprotinin.
Inhibitors by hepatic synthesis
These circulating factors include a number of serine protease inhibitors or serpins. This superfamily of proteins plays a major role in the regulation of coagulation, fibrinolysis, and inflammation. The serpins function as suicidal inhibitors, presenting their reactive centre as a pseudo-substrate for their target. Although hydrolysis is attempted by the protease it cannot be completed and a tight 1:1 complex is formed which is rapidly cleared from the circulation. For example, the half-life of the thrombin ATIII (TAT) complex is about 5 min. The two plasma inhibitors found most commonly are ATIII, which accounts for about 60% of plasma anticoagulant activity, and HCII which accounts for a further 30% of the total activity.
ATIII is synthesized in the liver and is not vitamin K-dependent. This inhibitor irreversibly neutralizes factors Xlla, Xla, IXa, Xa, and thrombin (Fig. 7). In vivo, glycosaminoglycans such as heparan sulphate on the endothelial cell surface are the initiators of the enhanced ATIII inhibitory function.53 Heparan is a varying chain length mucopolysaccharide or glycosaminoglycan that is tethered to the surface of the endothelium by a protein skeleton. Contact with this surface will induce a conformational change in ATIII. This combination produces the physiological effect of a vascular surface with profound anticoagulant properties.
ATIII has a biological half-life of 35 days and is produced at a relatively constant rate.14 It is not an acute phase respondent and so production does not change rapidly in response to stress. Deficiency of ATIII can be congenital or acquired. The normal range for ATIII is based on a comparison with pooled plasma and is quoted as 85120%. The congenital forms can be divided into those associated with an absence or reduction of ATIII in the plasma and those associated with an amino acid sequence that bestows inappropriate inhibitory activity to the molecule. Both these defects are associated with an increased risk of thromboembolic disease.
Acquired ATIII deficiency is seen in a number of states including certain chemotherapeutic regimens (L-asparaginase treatment), hepatic failure, nephrotic syndrome, severe pre-eclampsia, shock, disseminated intravascular coagulation (DIC), and after certain surgeries such as those involving extracorporeal circulation. ATIII deficiency is also seen in chronic heparin administration, to produce heparin resistance.
Pregnancy represents an interesting example and model of reduced ATIII activity that is relevant to other clinical arenas. Fibrinogen concentration and platelet count increase during pregnancy,57 and in pre-eclampsia there are diminished levels of ATIII. This fall in ATIII levels reflects a consumptive process as the plasma TAT complexes increase as ATIII levels drop.44
The level of ATIII that may be cause for concern has not been accurately defined. However, patients who have undergone shock and demonstrate levels of ATIII below 5060% of normal activity have an increased morbidity and mortality.7 24 54 85 Patients with levels below 20% have near 100% mortality.
Replacement or enhancement of ATIII concentrations has been suggested in a number of these conditions. Concentrates from human sources have been available for some time and have shown some benefits in patients with sepsis syndrome. A recombinant form of ATIII has entered clinical trials as a method of reducing heparin resistance in patients before heart surgery.
HCII is the second plasma thrombin inhibitor. The endothelial glycosaminoglycan, dermatan sulphate has a specific binding site for HCII.52 This binding site is a hexasaccharide without structural similarity with the pentasaccharide of heparan or heparin.
Therapies to inhibit thrombin production or activity
Included in this category are recombinant agents equivalent to some naturally occurring proteins and totally synthetic agents. A most important point to note is that at times these drug therapies will produce prothrombotic or hypercoagulable states. Warfarin therapy is associated with cutaneous thrombosis.71 73 This latter effect is a result of the action of warfarin to reduce effective protein C concentration (a vitamin K-dependent factor) and induce a prothrombotic protein C deficiency.30 Moreover, studies in patients given warfarin immediately after myocardial revascularization show a short period of a hypercoagulable state, due directly to the administration of warfarin.38 Heparin will induce a thrombotic state by direct or immune-mediated platelet activation as described later.
The first group of antithrombin drugs discussed are not direct inhibitors of thrombin but aim to slow thrombin generation and presentation.
Vitamin K antagonists
Reduction in clotting factor activity is produced when patients are given vitamin K antagonists. The first oral anticoagulant used was dicoumarol that was isolated from spoilt clover. This agent had a poor absorption and non-linear kinetics and is no longer used. The three widely used drugs are warfarin, phenprocouman, and acenocouman.
Warfarin is the best known of this class of agent and is used prophylactically in atrial fibrillation, venous thrombosis, pulmonary embolism, and in patients with prosthetic heart valves. The effect of warfarin is monitored by the PT or the International Normalized Ratio (INR). The INR was developed by the World Health Organization in the early 1980s to eliminate problems in oral anticoagulant therapy caused by variability in the sensitivity of different commercial sources and different batches of thromboplastin. The INR is derived by raising the observed ratio of PT in control and patient plasma to the power of an International Sensitivity Index (ISI). The ISI is a measure of the response to a thromboplastin preparation and is typically between 2 and 2.6 for most commercial rabbit-brain thromboplastins.30 The INR has for no obvious reason been only slowly adopted within North America compared with the rest of the international community. The PT ratio, which has been adopted by centres in North America, is not directly interchangeable with INR. This adds some confusion when discussing results from studies of the effects of an anticoagulant regimen on outcome. An INR of 2.5 is adequate for treating venous thrombosis, pulmonary thromboembolism and for atrial fibrillation. An INR of 3.5 is the target for patients with heart valves.1
Warfarin can be given intravenously but is usually given orally and is well absorbed. Peak plasma concentrations of warfarin occur about 90 min after ingestion. The plasma half-life is about 36 h and it usually takes about 3 days of daily dosing to achieve a steady-state concentration of the drug. A prolonged coagulation time requires more than a 25% decrease in factor activity. This takes about 824 h following ingestion of warfarin. The peak effect of a single dose occurs at 3672 h and lasts about 5 days.
Warfarin is a racemic mixture of about equal amounts of so-called R and S forms. The S form is about five times more potent as a vitamin K antagonist than the R form and is oxidized in the liver to hydroxywarfarin that is excreted in the bile. The R form is metabolized to warfarin alcohols, which are excreted by the kidney. About 97% of warfarin in the circulation is bound to albumin. Given the above confounding variables it is not surprising that the biological effect of warfarin to prolong the PT can be significantly altered by a multitude of other therapeutic interventions as shown in Table 2.
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In the context of the perioperative period, there are few evidence-based criteria to guide patient management. The risks of thromboembolism if the patient is not anticoagulated must be balanced with the risks of excessive intraoperative bleeding. For example, in a survey of patients having dental surgery, the incidence of significant bleeding (0.05%) was similar to those with thrombotic complications (0.03%).67 The major difference was that four of the five patients with thrombotic complications died as a consequence of these events. Although the likelihood of significant bleeding may be greater with more major procedures such as bowel or orthopaedic surgery, this must be balanced against the magnitude of the thrombotic risk and its effects. Although it is common practice to stop warfarin up to a week before surgery, and substitute the more readily reversible heparin, there is little current evidence to show this is an absolute necessity. This is especially the case in patients with prosthetic cardiac valves.15 One other recent review suggested that withholding warfarin for 48 h was associated with a 1 point fall in the INR from about 3.5 to 2.5 in patients with prosthetic valves. This value was not associated with increased bleeding or increased thrombotic episodes over the transient period of drug withdrawal.79
One further area of concern is the patient taking oral anticoagulants whom becomes pregnant. In particular, the management of women with prosthetic heart valves during pregnancy poses a particular challenge, as there are no available controlled clinical trials to provide guidelines for effective antithrombotic therapy. Warfarin is teratogenic and should not be given in the first trimester of pregnancy. However, subcutaneous (s.c.) administration of heparin is reported to be ineffective in preventing thromboembolic complications. A recent literature review12 suggested that the regimen associated with the lowest risk of valve thrombosis was the use of oral anticoagulants throughout pregnancy. Although this approach was associated with warfarin embryopathy in 6.4% (95% CI 4.68.9%) of live births, this was less than the 9.2% (95% CI 5.913.9) risk of valve thrombosis using heparin only between 6 and 12 weeks gestation.
Heparins
Heparin is a naturally occurring negatively charged sulphated polysaccharide with a complex structure. It is a glycosaminoglycan formed from alternating residues of D-glucosamine and L-iduronic acid. The important part of the molecule with regard to anticoagulation is thought to be a specific, ATIII binding, pentasaccharide sequence found in about one-third of molecules of UFH, with a lower proportion in molecules of the low-molecular weight product.32 33 Heparin is located mostly in mast cells in lungs, intestine, and liver in mammals. Heparin was originally isolated from liver during investigations to ascertain if the phospholipid component of cephalin would cause clotting. Since the discovery of heparin in 1916 by McLean,29 numerous physiological actions have been proposed for this agent. The finding of heparin-rich mast cells in tissues where the inside and outside of the body are in close proximity (the skin, lung, and gut) suggests a primary anti-inflammatory or immunological role for this agent. This concept is strengthened when we consider first, that heparin alone has no direct effects on coagulation, and secondly, is found in lower orders of the animals, such as molluscs, which lack a coagulation system.
UFH
Standard preparations of heparin are unfractionated (UFH), derived from either porcine intestine or bovine lung and prepared as either calcium or sodium salts. The number and sequence of the saccharides is variable, with molecular weights ranging from 3000 to 30 000 Da, with a mean of 15 000 Da representing 4050 saccharides in length. There is no apparent difference between any of the available forms of UFH with respect to their pharmacology or anticoagulant profile.31
Low-molecular weight heparin
Low-molecular weight heparins (LMWH) are produced from UFH by chemical or enzymatic depolymerization.21 82 This produces marked changes in the properties of the heparin and leads to the difference between the clinical effects, pharmacokinetics and pharmacodynamics of LMWH compared with UFH.31 33 LMWHs have mean molecular weights of 40006500 Da, although the range is 200010 000 Da. There are significant variations between the different commercial preparations according to the method used in their production as shown in Table 3.
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The augmentation of the inhibitory effect of ATIII comes about in three separate ways. First, heparin attaches to a small, high affinity site on ATIII to produce a conformational change at the reactive site (Fig. 8). This occurs with the pentasaccharide sequence alone and results in a 100-fold increase in inhibition of not only thrombin but also factor Xa and certain other proteases, including coagulation factors XIIa, XIa, IXa, plasmin, and kallikrein. Interestingly, loss of affinity for the pentasaccharide at this site (such as found in ATIII Geneva16 or ATIII Rouen62 that have point mutations of this site) leads to only a mild tendency to develop venous thrombosis,10 suggesting other effects of heparin on ATIII.
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The third effect is also specific to the inhibition of thrombin, which has charged exosites away from the active centre. One of these exosites attaches to longer (more than 18 residues) non-specific parts of the heparin molecule which are bound to the high affinity site by their pentasaccharide sequence.60 This long tail ensures a certain orientation of thrombin with antithrombin and fixes the antithrombin portion in a ternary complex with heparin and thrombin (Fig. 8). In this circumstance, the role of heparin is to bring together the protease and its inhibitor (a process termed approximation) rather than just producing the conformational change.59 This approximation and binding to the heparin chain are essential for the accelerated inhibition of thrombin by ATIII.46 In contrast, approximation plays little part in the inhibition of proteases that bind weakly to heparin such as factors IXa, Xa, and XIIa. These tend to be inhibited directly by the heparinATIII complex and approximation does not need to take place.5 31
The importance of chain length in relation to the range and specificity of the catalytic action of heparin can be best appreciated from studies of small, semi-synthetic oligosaccharides.75 These studies showed that with less than 18 saccharides, there was little activity to inhibit thrombin (as shown using the thrombin time coagulation test) compared with a 140-fold inhibition of this test with molecules with more than 18 residues. Increased factor Xa inhibition was detected with both chain lengths but was 23-fold more potent with lower numbers.
Following the reaction between the active site of the protease and the reactive site of ATIII, a further conformational change in ATIII occurs that causes it to envelop the protease. This change also reduces the affinity of ATIII for heparin, which is released to participate in further ATIII-protease reactions. Heparin has two further antithrombotic actions in addition to those mediated via ATIII.
First, heparin can activate the other major circulating antithrombin, HCII. This activation does not require the pentasaccharide sequence but does require heparins of greater than 7200 Da or 24 saccharide units in length. Activation of HCII is assumed to be a non-specific effect of heparin related to the total charge on the molecule rather than acting via a specific receptor. This non-specific activation may explain why the HCII-dependent effect requires a 10-fold higher concentration (typically >4 IU ml1) than that required to activate ATIII.76 Secondly, heparin will stimulate the release of TFPI, reducing prothrombinase production via the extrinsic pathway. Plasma concentrations of TFPI increase 26-fold following heparin injection. This increase occurs with UFH and LMWH.8
Pharmacokinetics and pharmacodynamics of heparins
Similar to the mechanism of action of UFH and LMWH, the pharmacokinetics and dynamics have a number of differences and some similarities. In particular, neither significantly cross the placenta. Moreover, the plasma concentrations of heparin are not uniformly related to the anticoagulant effect produced and there is a wide variability in doseresponse effects in patients.84
UFH
The pharmacokinetics of UFH are complex. Heparins are poorly absorbed from the gastrointestinal tract and can cause haematomas after intramuscular injection. They are therefore usually administered by s.c. or intravenous (i.v.) injection. I.v. injection is the preferred route when a rapid anticoagulant effect is needed. However, similar levels of anticoagulation can be achieved, with onset delayed by 1 or 2 h, by the s.c. route, if sufficient doses are used.31 Studies suggest the safety of the two routes is comparable.
The heterogeneity of heparin molecules produces great variability in the plasma concentration of the agent in relation to the dose administered. A three-compartment model best describes the kinetics of UFH in humans. After injection, plasma levels initially decline rapidly a result of redistribution and uptake by endothelial cells. More than 50% of heparin circulates bound to proteins including platelet factor 4, histidine-rich glycoprotein, vitronectin, fibronectin, and vWF. The first three of these also reduce its bioavailability and activity. Raised concentrations of these proteins may account for the heparin resistance seen in malignancy and inflammatory disorders.29 The release of platelet factor 4 from activated platelets may reduce heparin concentration at the site of clot formation, contributing to the reduced efficacy of heparin against clot-bound thrombin.
Heparin clearance is non-linear and elimination occurs by two separate processes; a rapid mechanism, which is readily saturated at clinically therapeutic concentrations, and a slower process involving first-order kinetics. The rapid, saturable phase of heparin clearance is thought to be a result of cellular degradation by macrophages, which internalize the heparin, then depolymerize and desulphate it. Saturation occurs when all the receptors have been utilized and further clearance depends on new receptor synthesis. This process explains the poor bioavailability of heparin after low-dose s.c. injection: the slow rate of absorption barely exceeding the capacity for cellular degradation. Significant plasma levels can only be achieved by saturation of these receptors with a loading dose. The slower phase of heparin elimination is a result of renal excretion. This complex mechanism of elimination means that as the dose of heparin is increased, the elimination half-life appears to increase in duration. A bolus of 25 unit kg1 has an apparent half-life of 30 min which increases to 60 min with a dose of 100 unit kg1 and this half-life duration is further increased to 150 min when the bolus dose is 400 unit kg1.6 17 Surprisingly, no consistent report of the effects of renal or hepatic dysfunction on the pharmacokinetics of heparin have been described.29 31
LMWH
The affinity of plasma proteins for LMWHs is much less than for UFH, so that only 10% is protein bound. Moreover, LMWHs are not subject to the rapid degradation that UFH suffers as they are not inactivated by platelet factor 4 and do not bind to endothelial cells or macrophages. This produces nearly complete bioavailability, compared with 40% for low-dose s.c. UFH, and this guarantees a more predictable anticoagulant action. They are almost completely absorbed following s.c. injection.
In contrast to UFH, the LMWHs exhibit linear pharmacokinetics with proportionality between anti-Xa (and anti-IIa in some cases) plasma concentration and dose. Their distribution volume is close to the blood volume. Similar to UFH they are partially metabolized by desulphation and depolymerization.
Although the clearance of LMWH is dependent on renal excretion, producing a half-life two to four times as long as UFH, this is not clinically relevant until there is severe renal disease and creatinine clearance values less than 15 ml min1 are achieved.35
Urinary excretion of anti-Xa activity for enoxaparin, dalteparin, and nadroparin, all given at doses for prevention of venous thrombosis, is between 3 and 10% of the injected dose. However, these LMWHs differ in the extent of their non-renal clearance, resulting in different apparent elimination half-life values and relative apparent bioavailability.
The LMWHs available differ in the distribution of molecular weights, in vitro potency, bioavailability, anti-Xa activity,22 and consequently their pharmacodynamic behaviour, recommended dose regimen, and efficacy/safety ratio.26 Because of these differences among LMWHs, the clinical profile of a given LMWH cannot be extrapolated to another one or generalized to the whole LMWH family. Table 4 shows data for potency, plasma half-life, and bioavailability for certain currently available agents.
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There is a marked variation in the initial anticoagulant response to a fixed dose of UFH.
Variability in saturation of cellular binding and degradation will enhance the non-linear response following repeat dosage.
The risk of recurrent thromboembolism is reduced if the effect of heparin is maintained above the lower therapeutic limit.37 48
Direct measurement of heparin concentration is not possible, although protamine titration can be used to ascertain blood levels. The commonly used laboratory test of heparin function is the aPTT, which measures the effect of heparin-activated coagulation inhibition. The therapeutic range most commonly quoted is an aPTT between 1.5 and 2.5 times the control value. However the commercially available kits for measurement of aPTT differ in their sensitivity to heparins. This suggests that the protamine titration method of monitoring may be more robust.9
The higher bioavailability of LMWH and their longer half-life produce a more predictable anticoagulant response. These properties allow once daily s.c. administration for prophylaxis of deep venous thrombosis.33 37 Although no laboratory monitoring is required for the majority of patients receiving LMWH, it is important to check anti-Xa activity in those who are renally impaired, as pregnancy progresses (with associated fluctuations in weight and plasma volume), and where thrombosis and/or haemorrhage occur or are suspected while on treatment. The anti-Xa assay used must also be calibrated against the brand of LMWH prescribed.
Side effects of heparin
Although haemorrhage is rare with prophylactic doses of either UFH or LMWH given alone, it is a frequent complication of therapeutic heparin administration. The greater the dose of heparin and therefore the greater its anticoagulant effect, the greater the risk of haemorrhage. When comparable doses are used, the risks are similar using either the continuous i.v. or s.c. route of administration.29 31 Many patient factors are known to increase the risk of haemorrhage including the duration of treatment, presence of cardiac, hepatic or renal dysfunction, aspirin or other platelet active therapy, recent surgery, and trauma or invasive procedures. There is, for example, evidence for increased, and more prolonged bleeding after emergency cardiac surgery in patients who have received platelet-active agents such as abciximab, clopidogrel, or ticlopidine,19 but not with agents such as eptifibatide20 which have a shorter duration of action.
Heparin will also impair platelet aggregation and inhibits platelet function by direct binding to platelets. It is the higher-molecular weight heparin molecules with the lower affinity for ATIII that interfere most with platelet function.68 These actions may be responsible for heparin-induced haemorrhage by a mechanism which is separate to its anticoagulant actions.
Approximately 30% of patients who suffer anticoagulant-related haemorrhage are found to have previously undiagnosed predisposing lesions, particularly of the gastrointestinal and genitourinary tracts. A review of this complication estimated the daily frequencies of fatal, major, and all types of haemorrhage in patients receiving therapeutic anticoagulation as 0.05, 0.8, and 2.0%, respectively, approximately twice the level expected in the absence of anticoagulation.45 The incidence of increased perioperative haemorrhage, which contributed to adverse outcome, has been reported variously in between 23 and 10%40 of patients receiving prophylactic heparin therapy.
Studies of LMWHs given for thromboprophylaxis suggest they cause an increase in wound haematomas but no change in the incidence of haemorrhage. In contrast, a significant reduction in major haemorrhage is seen when LMWHs are used to treat established thrombosis.33 48 Although chronic heparin administration has been associated with osteoporosis and hypoaldesteronism, these remain medical curiosities compared with the relatively common and potentially life-threatening problem of heparin-induced thrombocytopaenia (HIT) and especially that associated with thrombosis.
HIT occurs in about 2% of patients receiving therapeutic doses of heparin. Affected patients are generally receiving high doses of UFH given intravenously, but many cases have been reported in patients on low-dose s.c. heparin prophylaxis, and HIT has been attributed to flushing lines with heparin. The risk of HIT is less with LMWH than with UFH, perhaps because of the lesser interaction with platelets. Two distinct clinical syndromes have been described.13
Type I involves a mild thrombocytopaenia with a platelet count which rarely falls below 100x109 litre1, that occurs during the first few days of treatment and usually recovers rapidly even if heparin is continued. The patient is normally asymptomatic and no specific treatment is required. The underlying mechanism probably involves the action of heparin as a platelet aggregator.13 Type II HIT is characterized by a delayed onset of a severe, progressive thrombocytopaenia with a platelet count often below 50x109 litre1. The platelet count does not recover unless heparin therapy is stopped and recurs promptly if heparin is restarted. Recovery usually occurs within a week but may occasionally be prolonged. An immune mechanism has been suggested, in which heparin binds to platelet factor 4 to form a molecule which stimulates the production of an IgG antibody.80 This antibody binds the heparin-platelet factor 4 molecule to produce an immune complex, all three parts of which are capable of binding to platelets. These complexes have two separate effects. First, they coat platelets and increase their removal from the circulation by the reticuloendothelial system. Secondly, they cause activation of platelets and the coagulation cascade, leading to a hypercoagulable state.80 Haemorrhage is uncommon and resistance to anticoagulation may occur as a result of heparin-induced release of platelet factor 4. A high index of suspicion is necessary, as only immediate withdrawal of heparin will reduce mortality and morbidity. The condition is under diagnosed and should be considered in all patients receiving heparin who develop a new thrombosis or heparin resistance.13 80 Confirmation requires a platelet count to show thrombocytopaenia, a blood film to demonstrate clumping, exclusion of other causes for thrombocytopaenia and the presence of a heparin-dependent anti-platelet antibody. A rapid platelet aggregation test can be used to detect the antibody and confirm the diagnosis. The most serious complication associated with type II HIT is new thromboembolic events, due to platelet-rich thrombi, which continue to form until the heparin is withdrawn. Platelet counts should be monitored closely as precipitous falls may herald the onset of thrombosis even without absolute thrombocytopaenia. Arterial and venous thrombosis may occur either alone or together and multiple sites are often involved. In patients receiving therapeutic doses of porcine heparin, 0.4% exhibited manifestations of thrombosis, most commonly lower limb thrombosis, a thrombotic cerebrovascular accident or acute myocardial infarction.81 In one series of surgical patients the incidence was reported at 0.3%, but 80% (eight of 10) suffered major thromboembolic morbidity, five of these eight requiring limb amputation. In these circumstances, the conventional wisdom was to stop the heparin and commence warfarin as early as possible. However this approach has been questioned.
(a)It takes 35 days to reach a therapeutic level with oral anticoagulants and this period will need to be covered with an alternative antithrombotic agent. LMWHs have a high incidence of cross-reaction with the heparin-dependent antibody, which can be ascertained using the platelet aggregation test. If no cross-reaction occurs, successful anticoagulation can be safely undertaken. However, significant thrombotic events may have occurred before a change in therapy.47
(b)There is convincing evidence that during the early stages of warfarin administration, a therapy-related hypercoagulable state is produced. This is thought to contribute to the thrombotic complications of the underlying HIT.80
Two other therapies are available and licensed for use to provide anticoagulation in patients with HIT. The first is danaparoid sodium, which is a mixture of the endothelial cell glycosaminoglycans heparan, dermatan, and chondroitin sulphates. Effect is monitored using anti-Xa activity. The second agent is hirudin (Lepirudin) manufactured using recombinant technology. Use and monitoring of Lepirudin is discussed later.
Reversal of action: antidotes to heparins
The effects of UFH wear off so rapidly that an antagonist is rarely required, except after the high doses administered to facilitate cardiopulmonary bypass. Protamine is a basic protein extracted from fish sperm that combines with heparin to form a stable, inactive complex. Conventional wisdom was that equimolar amounts of protamine sulphate (1 mg protamine for 100 units of heparin) should be used to provide optimal neutralization. However, the use of protamine titration using various techniques has been shown to considerably reduce blood loss and transfusions after heart surgery using considerably lower doses of protamine.18 39 Although protamine will reduce the anticoagulant effects of heparin, it does not affect various other actions induced by heparin administration. Of relevance is the effect of heparin to increase plasma concentrations of free drug, such as diazepam and propanolol, is not inhibited or prevented by protamine administration.56
With LMWHs, protamine is able to neutralize the anti-IIa but not the anti-Xa action of heparin, because of inability to bind the smaller heparin molecules. An analogous situation occurs with the use of heparinase. This enzyme will break down the larger molecular weight UFH, but this will produce lower molecular weight compounds. This may be one reason for an increase in blood loss in patients who had heparin reversal with heparinase rather than with protamine.27 This effect may be extremely important in relation to local anaesthetic block.
Local anaesthesia in the patient receiving heparin
Therapeutic anticoagulation is a contraindication to central nerve block unless the coagulation profile is corrected to normal. The risks associated with epidural or spinal anaesthesia in patients receiving heparin prophylaxis is a controversial subject which has been the subject of a number of recent reviews.34 50 78
Reduced efficacy of heparin therapy
Although bound, thrombin can be inactivated by the heparinATIII complex, much higher concentrations are required than are needed to inactivate free thrombin.83 In addition, platelets secrete platelet factor 4, which neutralizes heparin. Clinically, this is seen as a requirement for much higher levels of heparin to prevent the extension of venous thrombosis compared with those required to prevent initiation of thrombosis.29 31 This poor efficacy of heparin is, in part, due to the fact that naturally occurring polypeptide thrombin inhibitors, such as ATIII, cannot inhibit bound thrombin.
Other agents used to inhibit thrombin
The inability of polypeptide inhibitors to directly target bound thrombin has been one reason for efforts to develop low-molecular weight, direct-acting, or site-directed, thrombin inhibitors. A further recurrent problem with the use of heparin and vitamin K antagonists is multiple sites of action. This leads to difficulties of accurate dosing and monitoring to maximize the therapeutic window and minimize the risk of bleeding. Finally, and as with nearly all of the drugs which effect the haemostatic or coagulation systems, the drive to produce newer, more specific agents, has largely come from studies in patients having interventional cardiology such as angioplasty or stent insertion. These agents are also of interest as they provide means to establish anticoagulation in the patient who has a history of type II heparin-induced thrombosis and thrombocytopaenia.
Hirudin
Hirudin is a polypeptide originally obtained from the medicinal leech. The molecule is now manufactured by recombinant technology with the generic name of Lepirudin. Two recombinant hirudin preparations, revasc (Novartis) and refludan (Aventis), are available for post-surgical DVT prophylaxis and alternate anticoagulant use in patients with heparin-induced thrombocytopenia.51 A synthetic antithrombin agent based on the combined structures of hirudin and antithrombin peptides, hirulog (Bivalirudin), is undergoing clinical trials in cardiovascular indications.2 Additional studies on the hirudins are being carried out to test their efficacy as anticoagulant replacements for heparin for both surgical and interventional cardiology indications.
Hirudin acts by irreversible binding to the active site of thrombin. The irreversibility is a result in part to the molecule having a long carboxyl tail which binds to the anion binding exosite of thrombin (Fig. 9).
|
The major clinical disadvantage of hirudin preparations is their mode of excretion by renal clearance leading to a plasma half-life of about 14 h. This duration is not shortened by dialysis or haemofiltration. Significant bleeding has been reported in patients having continuous venovenous haemofiltration and hirudin infusion,42 and also in patients having cardiac surgery.51 Unlike heparin or the vitamin K antagonists, there is no clinically proven antagonist for hirudin. Studies in animals have shown partial benefits to reduce bleeding using either activated prothrombin complex or recombinant factor VII in one report,23 or factor VIII and DDAVP in another.11 This is likely to prove one of the limiting factors for the optimal development of hirudin. One other problem is that patients have been reported to develop antibodies to the recombinant form,36 which have been associated with hypersensitivity reactions.
LMWH inhibitors
Arginine analogues
A number of chlormethyketones have been used in laboratory-based medicine as direct inhibitors of thrombin; however, they are too toxic for use in humans. Arginine is the amino acid residue at the active site of the serine protease inhibitors such as ATIII. A combination of these chemistries is found in TAME (tosyl-arginine methyl ester) which has formed the basis of two direct-acting inhibitors, argatroban and napsagatran.69 These small molecules are able to fit easily into the active pocket of thrombin and other serine protease and bind with great affinity (Fig. 9). Napsagatran has reached phase II studies in humans.
Argatroban has been extensively investigated and is the first clinically approved antithrombin agent. The molecular properties of argatroban (small, fast, selective, with reversible inhibition of the thrombin catalytic site, and similar in vitro potency for inhibiting both clot-bound and soluble thrombin) offer the potential for significant antithrombotic efficacy with minimal systemic anticoagulant effects. The i.v. agent, Novastan (a brand of argatroban), is currently approved for clinical use in Japan for the treatment of peripheral arterial occlusive disease, and in the USA for anticoagulation in patients with HIT and thrombosis. Novastan is in advanced clinical development in other countries for several indications, including therapy in heparin-induced thrombocytopenia and thrombosis syndrome, and as adjunctive therapy to thrombolytic agents in acute myocardial infarction.
The pharmacokinetic profile of argatroban is described by a two-compartment model with first-order elimination; mean (SD) clearance, steady-state volume of distribution, and half-life values from 40 healthy volunteers were 4.7 (1.1) ml min1 kg1, 179.5 (33.0) ml kg1, and 46.2 (10.2) min, respectively. Clearance is about 20% lower in the elderly. Kinetic analysis shows no significant differences in these variables in patients with renal dysfunction. With hepatic impairment, the maximum concentration and half-life of argatroban were increased approximately 2- to 3-fold, associated with a reduction in clearance to 25% of that in healthy volunteers.74 Control of anticoagulation is by the aPTT or ecarin clotting time. aPTT or ACT and plasma argatroban concentrations were well correlated. Dose regimens vary based on the clotting time but are typically in the range of 0.54 µg kg1 min1, as a continuous infusion for anticoagulation during haemofiltration or use of intra-aortic balloon counterpulsation, and about 2 µg kg1 min1 after a bolus of 100 µg kg1 for abdominal aortic surgery.58 These doses produce an ACT of about 180200 s.
Argatroban also appears to have certain other interesting effects that may increase its use and broaden its therapeutic indications. The first relates to the possibility that there is an increase in nitric oxide production associated with infusion of argatroban, which may prove useful in providing arteriolar dilatation in peripheral vascular disease.77 This nitric oxide effect may be a result of the presence of the arginine moiety in the molecule. The second aspect is related to effects on the cerebral circulation. A number of recent semi-anecdotal studies have suggested that ischaemic cerebral tissue can be revascularized and blood flow increased in humans during infusion of argatroban.41 43
Several of the synthetic thrombin inhibitors such as melagatran55 are also being developed for oral use.70 As the therapeutic index of thrombin inhibitors is narrower than that of heparin, this route may not be an optimal approach for the development of these agents. However, there is a potentially huge market for this class of compound, which could replace drugs such as heparin and warfarin in the next few years. Despite several unresolved developmental issues, the thrombin inhibitors provide an alternative to heparin anticoagulation and may prove to be useful in clinical use.
References
1 Anonymous. Guidelines on oral anticoagulation: third edition. Br J Haematol 1998; 101: 37487[Medline]
2 Anonymous. Effects of recombinant hirudin (lepirudin) compared with heparin on death, myocardial infarction, refractory angina, and revascularisation procedures in patients with acute myocardial ischaemia without ST elevation: a randomised trial. Organisation to Assess Strategies for Ischemic Syndromes (OASIS-2) Investigators. Lancet 1999; 353: 42938[ISI][Medline]
3 Adar R, Papa MZ, Amsterdam E, Bass A, Schneiderman J. Antithrombosis routines and hemorrhagic complications: a seven year survey comparing vascular and general surgical operations. J Cardiovasc Surg 1985; 26: 2759[ISI][Medline]
4 Bertina RM, Koeleman BP, Koster T, et al. Mutation in blood coagulation factor V associated with resistance to activated protein C. Nature 1994; 369: 6475[ISI][Medline]
5 Bjork I, Lindahl U. Mechanism of the anticoagulant action of heparin. Mol Cell Biochem 1982; 48: 16182[ISI][Medline]
6 Bjornsson TD, Wolfram KM, Kitchell BB. Heparin kinetics determined by three assay methods. Clin Pharmacol Ther 1982; 31: 10413[ISI][Medline]
7 Blauhut B, Kramar H, Vinazzer H, Bergmann H. Substitution of antithrombin III in shock and DIC: a randomized study. Thromb Res 1985; 39: 819[ISI][Medline]
8 Bregengaard C, Nordfang O, Ostergaard P, et al. Pharmacokinetics of full length and two-domain tissue factor pathway inhibitor in combination with heparin in rabbits. Thromb Haemost 1993; 70: 4547[ISI][Medline]
9
Brill Edwards P, Ginsberg JS, Johnston M, Hirsh J. Establishing a therapeutic range for heparin therapy. Ann Intern Med 1993; 119: 1049
10
Bruce D, Perry DJ, Borg JY, Carrell RW, Wardell MR. Thromboembolic disease due to thermolabile conformational changes of antithrombin Rouen-VI (187 AsnAsp). J Clin Invest 1994; 94: 226574[ISI][Medline]
11 Butler KD, Dolan SL, Talbot MD, Wallis RB. Factor VIII and DDAVP reverse the effect of recombinant desulphatohirudin (CGP 39393) on bleeding in the rat. Blood Coagul Fibrinolysis 1993; 4: 45964[ISI][Medline]
12
Chan WS, Anand S, Ginsberg JS. Anticoagulation of pregnant women with mechanical heart valves: a systematic review of the literature. Arch Intern Med 2000; 160: 1916
13 Chong BH. Heparin-induced thrombocytopenia. Br J Haematol 1995; 89: 4319[ISI][Medline]
14 Collen D, Schetz J, de Cock F, Holmer E, Verstraete M. Metabolism of antithrombin III (heparin cofactor) in man: effects of venous thrombosis and of heparin administration. Eur J Clin Invest 1977; 7: 2735[ISI][Medline]
15 Coulshed DS, Fitzpatrick MA, Lee CH. Drug treatment associated with heart valve replacement. Drugs 1995; 49: 897911[ISI][Medline]
16 de Moerloose PA, Reber G, Vernet P, Minazio P, Bouvier CA. Antithrombin III Geneva: a hereditary abnormal ATIII with defective heparin cofactor activity. Thromb Haemost 1987; 57: 1547[ISI][Medline]
17 de Swart CA, Nijmeyer B, Roelofs JM, Sixma JJ. Kinetics of intravenously administered heparin in normal humans. Blood 1982; 60: 12518[Abstract]
18
Despotis GJ, Joist JH, Hogue CW Jr, et al. The impact of heparin concentration and activated clotting time monitoring on blood conservation. A prospective, randomized evaluation in patients undergoing cardiac operation. J Thorac Cardiovasc Surg 1995; 110: 4654
19 Dyke C, Bhatia D. Inhibitors of the platelet receptor glycoprotein IIbIIIa and complications during percutaneous coronary revascularization. Management strategies for the cardiac surgeon. J Cardiovasc Surg 1999; 40: 50516[ISI][Medline]
20
Dyke CM, Bhatia D, Lorenz TJ, et al. Immediate coronary artery bypass surgery after platelet inhibition with eptifibatide: results from PURSUIT. Platelet glycoprotein IIb/IIIa in unstable angina: receptor suppression using integrelin therapy. Ann Thorac Surg 2000; 70: 86671
21 Fareed J, Hoppensteadt DA. Pharmacology of the low-molecular-weight heparins. Semin Thromb Hemost 1996; 22 (Suppl. 2): 1318[Medline]
22 Fareed J, Walenga JM, Hoppensteadt D, Huan X, Racanelli A. Comparative study on the in vitro and in vivo activities of seven low-molecular-weight heparins. Haemostasis 1988; 18 (Suppl. 3): 315[ISI][Medline]
23 Fareed J, Walenga JM, Pifarre R, Hoppensteadt D, Koza M. Some objective considerations for the neutralization of the anticoagulant actions of recombinant hirudin. Haemostasis 1991; 21 (Suppl. 1): 6472[ISI][Medline]
24 Fourrier F, Chopin C, Huart JJ, et al. Double-blind, placebo-controlled trial of antithrombin III concentrates in septic shock with disseminated intravascular coagulation. Chest 1993; 104: 8828[Abstract]
25 Francis CW, Markham RE Jr, Barlow GH, et al. Thrombin activity of fibrin thrombi and soluble plasmic derivatives. J Lab Clin Med 1983; 102: 22030[ISI][Medline]
26 Frydman A. Low-molecular-weight heparins: an overview of their pharmacodynamics, pharmacokinetics and metabolism in humans. Haemostasis 1996; 26 (Suppl. 2): 2438[ISI][Medline]
27 Gravlee G, Vester S, Regensburger D, et al. Heparinase-1 vs. protamine for heparin neutralization after bypass. Anesth Analg 2000; 90: SCA18(abstract)[ISI]
28
Hamamoto T, Yamamoto M, Nordfang O, et al. Inhibitory properties of full-length and truncated recombinant tissue factor pathway inhibitor (TFPI). Evidence that the third Kunitz-type domain of TFPI is not essential for the inhibition of factor VIIa-tissue factor complexes on cell surfaces. J Biol Chem 1993; 268: 870410
29 Hirsh J. Heparin. N Engl J Med 1991; 324: 156574[ISI][Medline]
30 Hirsh J. Oral anticoagulant drugs. N Engl J Med 1991; 324: 186575[ISI][Medline]
31 Hirsh J, Dalen JE, Deykin D, Poller L. Heparin: mechanism of action, pharmacokinetics, dosing considerations, monitoring, efficacy, and safety. Chest 1992; 102 (4 Suppl.): 337s51s[Medline]
32 Hirsh J, Levine MN. Low molecular weight heparin. Blood 1992; 79: 117[ISI][Medline]
33 Hirsh J, Levine MN. Low molecular weight heparin: laboratory properties and clinical evaluation. A review. Eur J Surg Suppl 1994; 571: 922[Medline]
34 Horlocker TT, Wedel DJ. Anticoagulation and neuraxial block: historical perspective, anesthetic implications, and risk management. Reg Anesth Pain Med 1998; 23 (6 Suppl. 2): 12934[ISI]
35 Hory B, Claudet MH, Magnette J, Bechtel P, Bayrou B. Pharmacokinetics of a very low molecular weight heparin in chronic renal failure. Thromb Res 1991; 63: 3117[ISI][Medline]
36 Huhle G, Hoffmann U, Song X, et al. Immunologic response to recombinant hirudin in HIT type II patients during long-term treatment. Br J Haematol 1999; 106: 195201[ISI][Medline]
37 Hull RD, Pineo GF. Low molecular weight heparin treatment of venous thromboembolism. Prog Cardiovasc Dis 1994; 37: 718[ISI][Medline]
38 Iguchi A, Sato K. Protein C response to induction of warfarin treatment after coronary bypass operation. Thorac Cardiovasc Surg 1994; 42: 2224[ISI][Medline]
39
Jobes DR, Aitken GL, Shaffer GW. Increased accuracy and precision of heparin and protamine dosing reduces blood loss and transfusion in patients undergoing primary cardiac operations. J Thorac Cardiovasc Surg 1995; 110: 3645
40 Kakkar VV, Boeckl O, Boneu B, et al. Efficacy and safety of a low-molecular-weight heparin and standard unfractionated heparin for prophylaxis of postoperative venous thromboembolism: European multicenter trial. World J Surg 1997; 21: 28[ISI][Medline]
41
Kario K, Matsuo T, Hoshide S, Umeda Y, Shimada K. Effect of thrombin inhibition in vascular dementia and silent cerebrovascular disease. An MR spectroscopy study. Stroke 1999; 30: 10337
42 Kern H, Ziemer S, Kox WJ. Bleeding after intermittent or continuous r-hirudin during CVVH. Intensive Care Med 1999; 25: 13114[ISI][Medline]
43 Kobayashi S, Tazaki Y. Effect of the thrombin inhibitor argatroban in acute cerebral thrombosis. Semin Thromb Hemost 1997; 23: 5314[Medline]
44 Kobayashi T, Tokunaga N, Sugimura M, et al. Coagulation/fibrinolysis disorder in patients with severe preeclampsia. Semin Thromb Hemost 1999; 25: 4514[ISI][Medline]
45 Landefeld CS, Beyth RJ. Anticoagulant-related bleeding: clinical epidemiology, prediction, and prevention [see comments]. Am J Med 1993; 95: 31528[ISI][Medline]
46 Lane DA, Denton J, Flynn AM, Thunberg L, Lindahl U. Anticoagulant activities of heparin oligosaccharides and their neutralization by platelet factor 4. Biochem J 1984; 218: 72532[ISI][Medline]
47 Leroy J, Leclerc MH, Delahousse B, et al. Treatment of heparin-associated thrombocytopenia and thrombosis with low molecular weight heparin (CY 216). Semin Thromb Hemost 1985; 11: 3269[ISI][Medline]
48 Litin SC, Gastineau DA. Current concepts in anticoagulant therapy. Mayo Clin Proc 1995; 70: 26672[ISI][Medline]
49 Liu CY, Nossel HL, Kaplan KL. The binding of thrombin by fibrin. J Biol Chem 1979; 254: 104215[Medline]
50 Liu S, Carpenter R, Neal J. Epidural anesthesia and analgesia: their role in postoperative outcome. Anesthesiology 1995; 82: 1474506[ISI][Medline]
51
Longrois D, de Maistre E, Bischoff N, et al. Recombinant hirudin anticoagulation for aortic valve replacement in heparin-induced thrombocytopenia. Can J Anaesth 2000; 47: 25560
52
Maimone MM, Tollefsen DM. Structure of a dermatan sulfate hexasaccharide that binds to heparin cofactor II with high affinity. J Biol Chem 1990; 265: 1826371
53 Marcum JA, Rosenberg RD. Anticoagulantly active heparin-like molecules from vascular tissue. Biochemistry 1984; 23: 17307[ISI][Medline]
54 Massignon D, Lepape A, Bienvenu J, et al. Coagulation/fibrinolysis balance in septic shock related to cytokines and clinical state. Haemostasis 1994; 24: 3648[ISI][Medline]
55 Mehta JL, Chen L, Nichols WW, et al. Melagatran, an oral active-site inhibitor of thrombin, prevents or delays formation of electrically induced occlusive thrombus in the canine coronary artery. J Cardiovasc Pharmacol 1998; 31: 34551[ISI][Medline]
56 Naranjo CA, Khouw V, Sellers EM. Nonfatty acid-modulated variations in drug binding due to heparin. Clin Pharmacol Ther 1982; 31: 74652[ISI][Medline]
57 Nilsson IM, Kullander S. Coagulation and fibrinolytic studies during pregnancy. Acta Obstet Gynecol Scand 1967; 46: 27385[ISI][Medline]
58 Ohteki H, Furukawa K, Ohnishi H, et al. Clinical experience of argatroban for anticoagulation in cardiovascular surgery. Jpn J Thorac Cardiovasc Surg 2000; 48: 3946[Medline]
59
Olson ST, Bjork I. Predominant contribution of surface approximation to the mechanism of heparin acceleration of the antithrombin-thrombin reaction. Elucidation from salt concentration effects. J Biol Chem 1991; 266: 635364
60
Olson ST, Bjork I, Sheffer R, et al. Role of the antithrombin-binding pentasaccharide in heparin acceleration of antithrombin-proteinase reactions. Resolution of the antithrombin conformational change contribution to heparin rate enhancement. J Biol Chem 1992; 267: 1252838
61
Olson ST, Srinivasan KR, Bjork I, Shore JD. Binding of high affinity heparin to antithrombin III. Stopped flow kinetic studies of the binding interaction. J Biol Chem 1981; 256: 110739
62 Owen MC, Borg JY, Soria C, et al. Heparin binding defect in a new antithrombin III variant: Rouen, 47 Arg to His. Blood 1987; 69: 12759[Abstract]
63 Potzsch B, Hund S, Madlener K, Unkrig C, Muller-Berghaus G. Monitoring of recombinant hirudin: assessment of a plasma-based ecarin clotting time assay. Thromb Res 1997; 86: 37383[ISI][Medline]
64 Potzsch B, Madlener K, Seelig C, et al. Monitoring of r-hirudin anticoagulation during cardiopulmonary bypassassessment of the whole blood ecarin clotting time. Thromb Haemost 1997; 77: 9205[ISI][Medline]
65 Rapaport SI. Regulation of the tissue factor pathway. Ann N Y Acad Sci 1991; 614: 5162[ISI][Medline]
66
Rosendaal FR, Siscovick DS, Schwartz SM, et al. Factor V Leiden (resistance to activated protein C) increases the risk of myocardial infarction in young women. Blood 1997; 89: 281721
67 Russo G, Corso LD, Biasiolo A, Berengo M, Pengo V. Simple and safe method to prepare patients with prosthetic heart valves for surgical dental procedures. Clin Appl Thromb Hemost 2000; 6: 903[ISI][Medline]
68 Salzman EW, Rosenberg RD, Smith MH, Lindon JN, Favreau L. Effect of heparin and heparin fractions on platelet aggregation. J Clin Invest 1980; 65: 6473[ISI][Medline]
69 Sanderson PE. Small, noncovalent serine protease inhibitors. Med Res Rev 1999; 19: 17997[ISI][Medline]
70 Sanderson PE, Lyle TA, Cutrona KJ, et al. Efficacious, orally bioavailable thrombin inhibitors based on 3-aminopyridinone or 3-aminopyrazinone acetamide peptidomimetic templates. J Med Chem 1998; 41: 446674[ISI][Medline]
71 Schramm W, Spannagl M, Bauer KA, et al. Treatment of coumarin-induced skin necrosis with a monoclonal antibody purified protein C concentrate. Arch Dermatol 1993; 129: 7536[Abstract]
72 Shore JD, Olson ST, Craig PA, Choay J, Bjork I. Kinetics of heparin action. Ann N Y Acad Sci 1989; 556: 7580[ISI][Medline]
73 Soundararajan R, Leehey DJ, Yu AW, Ing TS, Miller JB. Skin necrosis and protein C deficiency associated with vitamin K depletion in a patient with renal failure. Am J Med 1992; 93: 46770[ISI][Medline]
74 Swan SK, Hursting MJ. The pharmacokinetics and pharmacodynamics of argatroban: effects of age, gender, and hepatic or renal dysfunction. Pharmacotherapy 2000; 20: 31829[ISI][Medline]
75 Thomas DP, Merton RE, Barrowcliffe TW, Thunberg L, Lindahl U. Effects of heparin oligosaccharides with high affinity for antithrombin III in experimental venous thrombosis. Thromb Haemost 1982; 47: 2448[ISI][Medline]
76
Tollefsen DM, Majerus DW, Blank MK. Heparin cofactor II. Purification and properties of a heparin-dependent inhibitor of thrombin in human plasma. J Biol Chem 1982; 257: 21629
77 Ueki Y, Matsumoto K, Kizaki Y, et al. Argatroban increases nitric oxide levels in patients with peripheral arterial obstructive disease: placebo-controlled study. J Thromb Thrombolysis 1999; 8: 1317[ISI][Medline]
78 Vandermeulen EP, Van Aken H, Vermylen J. Anticoagulants and spinal-epidural anesthesia. Anesth Analg 1994; 79: 116577[ISI][Medline]
79 Wahl MJ. Myths of dental surgery in patients receiving anticoagulant therapy. J Am Dent Assoc 2000; 131: 7781[ISI][Medline]
80 Warkentin TE. Heparin-induced thrombocytopenia: a ten-year retrospective. Annu Rev Med 1999; 50: 12947[ISI][Medline]
81 Warkentin TE, Kelton JG. Heparin-induced thrombocytopenia. Annu Rev Med 1989; 40: 3144[ISI][Medline]
82
Weitz JI. Low-molecular-weight heparins. N Engl J Med 1997; 337: 68898
83 Weitz JI, Hudoba M, Massel D, Maraganore J, Hirsh J. Clot-bound thrombin is protected from inhibition by heparin-antithrombin III but is susceptible to inactivation by antithrombin III-independent inhibitors. J Clin Invest 1990; 86: 38591[ISI][Medline]
84 Whitfield LR, Schentag JJ, Levy G. Relationship between concentration and anticoagulant effect of heparin in plasma of hospitalized patients: magnitude and predictability of interindividual differences. Clin Pharmacol Ther 1982; 32: 50316[ISI][Medline]
85 Wilson RF, Mammen EF, Robson MC, et al. Antithrombin, prekallikrein, and fibronectin levels in surgical patients. Arch Surg 1986; 121: 63540[Abstract]