Platelets provide the initial hemostatic plug at sites of vascular injury. They also participate in pathological thromboses that lead to myocardial infarction, stroke, and peripheral vascular thromboses. Potent inhibitors of platelet function have been developed in recent years. These drugs act by discrete mechanisms, and thus in combination their effects are additive or even synergistic. Their availability has led to a revolution in cardiovascular medicine, whereby angioplasty and vascular stenting of lesions now is feasible with low rates of restenosis and thrombosis when effective platelet inhibition is employed.
Aspirin. Processes including thrombosis, inflammation, wound healing, and allergy are modulated by oxygenated metabolites of arachidonate and related polyunsaturated fatty acids that are collectively termed eicosanoids. Interference with the synthesis of eicosanoids is the basis for the effects of many therapeutic agents, including analgesics, antiinflammatory drugs, and antithrombotic agents
In platelets, the major cyclooxygenase product is thromboxane A2, a labile inducer of platelet aggregation and a potent vasoconstrictor. Aspirin blocks production of thromboxane A2 by acetylating a serine residue near the active site of platelet cyclooxygenase (COX-1), the enzyme that produces the cyclic endoperoxide precursor of thromboxane A2. Since platelets do not synthesize new proteins, the action of aspirin on platelet cyclooxygenase is permanent, lasting for the life of the platelet (7 to 10 days). Thus, repeated doses of aspirin produce a cumulative effect on platelet function. Complete inactivation of platelet COX-1 is achieved when 160 mg of aspirin is taken daily. Therefore, aspirin is maximally effective as an antithrombotic agent at doses much lower than those required for other actions of the drug. Numerous trials indicate that aspirin, when used as an antithrombotic drug, is maximally effective at doses of 50 to 320 mg per day (Antithrombotic Trialists' Collaboration, 2002; Patrono et al., 2004). Higher doses do not improve efficacy; moreover, they potentially are less efficacious because of inhibition of prostacyclin production, which can be largely spared by using lower doses of aspirin. Higher doses also increase toxicity, especially bleeding.
Other NSAIDs that are reversible inhibitors of COX-1 have not been shown to have antithrombotic efficacy and in fact may even interfere with low-dose aspirin regimens.
Dipyridamole. Dipyridamole (PERSANTINE) is a vasodilator that, in combination with warfarin, inhibits embolization from prosthetic heart valves. Dipyridamole has little or no benefit as an antithrombotic drug. In trials in which a regimen of dipyridamole plus aspirin was compared with aspirin alone, dipyridamole provided no additional beneficial effect (Antithrombotic Trialists' Collaboration, 2002). A single study suggests that dipyridamole plus aspirin reduces strokes in patients with prior strokes or transient ischemic attack (Diener et al., 1996). A formulation containing 200 mg of dipyridamole, in an extended-release form, and 25 mg of aspirin (AGGRENOX) is available. Dipyridamole interferes with platelet function by increasing the cellular concentration of adenosine 3¢,5¢-monophosphate (cyclic AMP). This effect is mediated by inhibition of cyclic nucleotide phosphodiesterase and/or by blockade of uptake of adenosine, which acts at adenosine A2 receptors to stimulate platelet adenylyl cyclase. The only current recommended use of dipyridamole is in combination with warfarin for postoperative primary prophylaxis of thromboemboli in patients with prosthetic heart valves.
Ticlopidine. Purinergic receptors respond to extracellular nucleotides as agonists. Platelets contain two purinergic receptors, P2Y1 and P2Y12; both are GPCRs for ADP. The ADP-activated platelet P2Y1 receptor couples to the Gq-PLC-IP3-Ca2+ pathway and induces a shape change and aggregation. The P2Y12 receptor couples to Gi and, when activated by ADP, inhibits adenylyl cyclase, resulting in lower levels of cyclic AMP and thereby less cyclic AMP-dependent inhibition of platelet activation. Based on pharmacological studies, it appears that both receptors must be stimulated to result in platelet activation (Jin and Kunapuli, 1998), and inhibition of either receptor is sufficient to block platelet activation. Ticlopidine (TICLID) is a thienopyridine that inhibits the P2Y12 receptor. Ticlopidine is a prodrug that requires conversion to the active thiol metabolite by a hepatic cytochrome P450 enzyme (Savi et al., 2000). It is rapidly absorbed and highly bioavailable. It permanently inhibits the P2Y12 receptor by forming a disulfide bridge between the thiol on the drug and a free cysteine residue in the extracellular region of the receptor and thus has a prolonged effect. Like aspirin it has a short half-life with a long duration of action, which has been termed "hit-and-run pharmacology" (Hollopeter et al., 2001). Maximal inhibition of platelet aggregation is not seen until 8 to 11 days after starting therapy. Thus, "loading doses" of 500 mg sometimes are given to achieve a more rapid onset of action. The usual dose is 250 mg twice per day. Inhibition of platelet aggregation persists for a few days after the drug is stopped.
Adverse Effects. The most common side effects are nausea, vomiting, and diarrhea. The most serious is severe neutropenia (absolute neutrophil count [ANC] <1500/mL), which occurred in 2.4% of stroke patients given the drug during premarketing clinical trials. Fatal agranulocytosis with thrombopenia has occurred within the first 3 months of therapy; therefore, frequent blood counts should be obtained during the first few months of therapy, with immediate discontinuation of therapy should cell counts decline. Platelet counts also should be monitored, as thrombocytopenia has been reported. Rare cases of thrombotic thrombocytopenic purpura-hemolytic uremic syndrome (TTP-HUS) have been associated with ticlopidine with a reported incidence of 1 in 1600 to 4800 patients when the drug is used after cardiac stenting; the mortality associated with these cases is reported to be as high as 18% to 57% (Bennett et al., 1998; Bennett et al., 1999). Remission of TTP has been reported when the drug is stopped (Quinn and Fitzgerald, 1999).
Therapeutic Uses. Ticlopidine has been shown to prevent cerebrovascular events in secondary prevention of stroke and is at least as good as aspirin in this regard (Patrono et al., 1998). It also reduces cardiac events in patients with unstable angina; however, its only FDA-approved indication is to reduce the risk of thrombotic stroke in patients who have experienced stroke precursors, and in patients who have had a completed thrombotic stroke. Since ticlopidine has a mechanism of action distinct from that of aspirin, combining the drugs might be expected to provide additive or even synergistic effects. This appears to be the case, and the combination has been used in patients undergoing angioplasty and stenting for coronary artery disease, with a very low frequency of stent thrombosis occurring over a short, 30-day follow-up (<1%) (Leon et al., 1998). As ticlopidine is associated with life-threatening blood dyscrasias and a relatively high rate of TTP, it is generally reserved for patients who are intolerant or allergic to aspirin or who have failed aspirin therapy.
Clopidogrel. The thienopyridine clopidogrel (PLAVIX) is closely related to ticlopidine and appears to have a slightly more favorable toxicity profile with less frequent thrombocytopenia and leukopenia, although thrombotic thrombocytopenic purpura has been reported (Bennett et al., 2000). Clopidogrel is a prodrug with a slow onset of action. The usual dose is 75 mg per day with or without an initial loading dose of 300 mg. The drug is equivalent to aspirin in the secondary prevention of stroke, and in combination with aspirin it appears to be as effective as ticlopidine and aspirin. It is used with aspirin after angioplasty and should be continued for at least 1 year (Steinhubl et al., 2002). In one study, the combination of clopidogrel and aspirin clearly was superior to aspirin alone; this finding suggests that the actions of the two drugs are synergistic, as might be expected from their distinct mechanisms of action (Yusuf et al., 2001). The FDA-approved indications for clopidogrel are to reduce the rate of stroke, MI, and death in patients with recent myocardial infarction or stroke, established peripheral arterial disease, or acute coronary syndrome.
Glycoprotein IIb/IIIa Inhibitors. Glycoprotein IIb/IIIa is a platelet-surface integrin which, by the integrin nomenclature, is designated aIIbb3. This dimeric glycoprotein is a receptor for fibrinogen and von Willebrand factor, which anchor platelets to foreign surfaces and to each other, thereby mediating aggregation. The integrin heterodimer/receptor is activated by platelet agonists such as thrombin, collagen, or thromboxane A2 to develop binding sites for its ligands, which do not bind to resting platelets. Inhibition of binding to this receptor blocks platelet aggregation induced by any agonist. Thus, inhibitors of this receptor are potent antiplatelet agents that act by a mechanism distinct from that of aspirin or the thienopyridine platelet inhibitors. Three agents are approved for use at present, with others under development.
Abciximab. Abciximab (REOPRO) is the Fab fragment of a humanized monoclonal antibody directed against the aIIbb3 receptor. It also binds to the vitronectin receptor on platelets, vascular endothelial cells, and smooth muscle cells. The antibody is used in conjunction with percutaneous angioplasty for coronary thromboses, and when used in conjunction with aspirin and heparin, has been shown to be quite effective in preventing restenosis, recurrent myocardial infarction, and death. The reduction in total events is about 50% in various large trials (Scarborough et al., 1999). The unbound antibody is cleared from the circulation with a half-life of about 30 minutes, but antibody remains bound to the aIIbb3 receptor and inhibits platelet aggregation as measured in vitro for 18 to 24 hours after infusion is stopped. It is given as a 0.25-mg/kg bolus followed by 0.125 mg/kg per minute for 12 hours or longer.
Adverse Effects.. The frequency of major hemorrhage in clinical trials varies from 1% to 10%, depending on the intensity of anticoagulation with heparin. Thrombocytopenia of less than 50,000 m/L is seen in about 2% of patients and may be due to development of neo-epitopes induced by bound antibody. Since the duration of action is long, if major bleeding or emergent surgery occurs, platelet transfusions can reverse the aggregation defect, because free antibody concentrations fall rapidly after cessation of infusion. Readministration of antibody has been performed in a small number of patients without evidence of decreased efficacy or allergic reactions. The expense of the antibody limits its use.
Eptifibatide. Eptifibatide (INTEGRILIN) is a cyclic peptide inhibitor of the fibrinogen binding site on aIIbb3. It blocks platelet aggregation in vitro after intravenous infusion into patients. Eptifibatide is given as a bolus of 180 mg/kg followed by 2 mg/kg per minute for up to 96 hours. It is used to treat acute coronary syndrome and for angioplastic coronary interventions. In the latter case, myocardial infarction and death have been reduced by about 20%. Although the drug has not been compared directly to abciximab, it appears that its benefit is somewhat less than that obtained with the antibody, perhaps because eptifibatide is specific for aIIbb3 and does not react with the vitronectin receptor. The duration of action of the drug is relatively short and platelet aggregation is restored within 6 to 12 hours after cessation of infusion. Eptifibatide generally is administered in conjunction with aspirin and heparin.
Adverse Effects. The major side effect is bleeding, as is the case with abciximab. The frequency of major bleeding in trials was about 10%, compared with about 9% in a placebo group, which included heparin. Thrombocytopenia has been seen in 0.5% to 1% of patients.
Tirofiban. Tirofiban (AGGRASTAT) is a nonpeptide, small-molecule inhibitor of aIIbb3 that appears to have a similar mechanism of action as eptifibatide. Tirofiban has a short duration of action and has efficacy in non-Q-wave myocardial infarction and unstable angina. Reductions in death and myocardial infarction have been about 20% compared to placebo, results similar to those with eptifibatide. Side effects also are similar to those of eptifibatide. The agent is specific to aIIbb3 and does not react with the vitronectin receptor. Meta-analysis of trials using aIIbb3 inhibitors suggests that their value in antiplatelet therapy after acute myocardial infarction is limited (Boersma et al., 2002). Tirofiban is administered intravenously at an initial rate of 0.4 mg/kg per minute for 30 minutes, and then continued at 0.1 mg/kg per minute for 12 to 24 hours after angioplasty or atherectomy. It is used in conjunction with heparin.
Philip W. Majerus and Douglas M. Tollefsen
key words: ANTIPLATELET, DRUGS, Aspirin
Tuesday, June 30, 2009
ANTIPLATELET DRUGS
Oral Anticoagulants
Phenprocoumon and Acenocoumarol. These agents are not generally available in the United States but are prescribed in Europe and elsewhere. Phenprocoumon (MARCUMAR) has a longer plasma half-life (5 days) than warfarin, as well as a somewhat slower onset of action and a longer duration of action (7 to 14 days). It is administered in daily maintenance doses of 0.75 to 6 mg. By contrast, acenocoumarol (SINTHROME) has a shorter half-life (10 to 24 hours), a more rapid effect on the PT, and a shorter duration of action (2 days). The maintenance dose is 1 to 8 mg daily.
Indandione Derivatives. Anisindione (MIRADON) is available for clinical use in some countries. It is similar to warfarin in its kinetics of action; however, it offers no clear advantages and may have a higher frequency of untoward effects. Phenindione (DINDEVAN) still is available in some countries. Serious hypersensitivity reactions, occasionally fatal, can occur within a few weeks of starting therapy with this drug, and its use can no longer be recommended.
Rodenticides. Bromadiolone, brodifacoum, diphenadione, chlorophacinone, and pindone are long-acting agents (prolongation of the PT may persist for weeks). They are of interest because they sometimes are agents of accidental or intentional poisoning. In this setting, reversal of the coagulopathy can require very large doses of vitamin K (i.e., >100 mg/day) for weeks or months.
Ximelagatran. Ximelagatran is a novel drug that is readily absorbed after oral administration and is rapidly metabolized to melagatran, a direct thrombin inhibitor. Therefore, its onset of action is much faster than that of warfarin. Ximelagatran is administered twice daily at a fixed dose and does not appear to require coagulation monitoring. Melagatran is excreted primarily by the kidney; therefore, dosage reduction may be necessary for patients with renal failure. Ximelagatran has been used successfully in clinical trials for prevention of venous thromboembolism (Francis et al., 2003; Schulman et al., 2003). Ximelagatran causes elevation of hepatic transaminases in about 6% of patients, but this side effect usually is asymptomatic and often is transient. The drug has not yet been approved for use in the United States.
Philip W. Majerus and Douglas M. Tollefsen
key words: blood, Oral Anticoagulants, Phenprocoumon and Acenocoumarol
PARENTERAL ANTICOAGULANTS
Heparin
Biochemistry. Heparin is a glycosaminoglycan found in the secretory granules of mast cells. It is synthesized from UDP-sugar precursors as a polymer of alternating D-glucuronic acid and N-acetyl-D-glucosamine residues (Sugahara and Kitagawa, 2002). About 10 to 15 glycosaminoglycan chains, each containing 200 to 300 monosaccharide units, are attached to a core protein and yield a proteoglycan with a molecular mass of 750,000 to 1,000,000 daltons. The glycosaminoglycan then undergoes a series of modifications, which include the following: N-deacetylation and N-sulfation of glucosamine residues, epimerization of D-glucuronic acid to L-iduronic acid, O-sulfation of iduronic and glucuronic acid residues at the C2 position, and O-sulfation of glucosamine residues at the C3 and C6 positions. Each of these modifications is incomplete, yielding a variety of oligosaccharide structures. After the heparin proteoglycan has been transported to the mast cell granule, an endo-b-D-glucuronidase degrades the glycosaminoglycan chains to fragments of 5000 to 30,000 daltons (mean, about 12,000 daltons or 40 monosaccharide units) over a period of hours.
Heparan Sulfate. Heparan sulfate is synthesized from the same repeating disaccharide precursor (D-glucuronic acid linked to N-acetyl-D-glucosamine) as is heparin. However, heparan sulfate undergoes less modification of the polymer than does heparin and therefore contains higher proportions of glucuronic acid and N-acetylglucosamine and fewer sulfate groups. Heparan sulfate on the surface of vascular endothelial cells or in the subendothelial extracellular matrix interacts with circulating antithrombin (see below) to provide a natural antithrombotic mechanism. Patients with malignancies may experience bleeding related to circulating heparan sulfate or related glycosaminoglycans that probably originate from lysis of the tumor cells.
Source. Heparin is commonly extracted from porcine intestinal mucosa or bovine lung, and preparations may contain small amounts of other glycosaminoglycans. Despite the heterogeneity in composition among different commercial preparations of heparin, their biological activities are similar (about 150 USP units/mg). The USP unit is the quantity of heparin that prevents 1 ml of citrated sheep plasma from clotting for 1 hour after the addition of 0.2 ml of 1% CaCl2.
Low-molecular-weight heparins (1000 to 10,000 daltons; mean, 4500 daltons, or 15 monosaccharide units) are isolated from standard heparin by gel filtration chromatography, precipitation with ethanol, or partial depolymerization with nitrous acid and other chemical or enzymatic reagents. Low-molecular-weight heparins differ from standard heparin and from each other in their pharmacokinetic properties and mechanism of action (see below). The biological activity of low-molecular-weight heparin is generally measured with a factor Xa inhibition assay, which is mediated by antithrombin (see below).
Mechanism of Action. Heparin catalyzes the inhibition of several coagulation proteases by antithrombin, a glycosylated, single-chain polypeptide composed of 432 amino acid residues (Olson and Chuang, 2002). Antithrombin is synthesized in the liver and circulates in plasma at an approximate concentration of 2.6 mM. It inhibits activated coagulation factors of the intrinsic and common pathways, including thrombin, Xa, and IXa; however, it has relatively little activity against factor VIIa. Antithrombin is a "suicide substrate" for these proteases; inhibition occurs when the protease attacks a specific Arg-Ser peptide bond in the reactive site of antithrombin and becomes trapped as a stable 1:1 complex.
Heparin increases the rate of the thrombin-antithrombin reaction at least a thousandfold by serving as a catalytic template to which both the inhibitor and the protease bind. Binding of heparin also induces a conformational change in antithrombin that makes the reactive site more accessible to the protease. Once thrombin has become bound to antithrombin, the heparin molecule is released from the complex. The binding site for antithrombin on heparin is a specific pentasaccharide sequence that contains a 3-O-sulfated glucosamine residue. This structure occurs in about 30% of heparin molecules and less abundantly in heparan sulfate. Other glycosaminoglycans (e.g., dermatan sulfate, chondroitin-4-sulfate, and chondroitin-6-sulfate) lack the antithrombin-binding structure and do not stimulate antithrombin. Heparin molecules containing fewer than 18 monosaccharide units (<5400 daltons) also do not catalyze inhibition of thrombin by antithrombin. Molecules of 18 monosaccharides or greater are required to bind thrombin and antithrombin simultaneously. In this case, catalysis may occur solely by induction of a conformational change in antithrombin that facilitates reaction with the protease. Low-molecular-weight heparin preparations produce an anticoagulant effect mainly through inhibition of Xa by antithrombin, because the majority of molecules are of insufficient length to catalyze inhibition of thrombin.
When the concentration of heparin in plasma is 0.1 to 1 units/ml, thrombin, factor IXa, and factor Xa are inhibited rapidly (half-lives less than 0.1 second) by antithrombin. This effect prolongs both the aPTT and the thrombin time (i.e., the time required for plasma to clot when exogenous thrombin is added); the PT is affected to a lesser degree. Factor Xa bound to platelets in the prothrombinase complex and thrombin bound to fibrin are both protected from inhibition by antithrombin in the presence of heparin. Thus, heparin may promote inhibition of factor Xa and thrombin only after they have diffused away from these binding sites. Platelet factor 4, released from the a-granules during platelet aggregation, blocks binding of antithrombin to heparin or heparan sulfate and may promote local clot formation at the site of hemostasis.
Miscellaneous Pharmacological Effects. High doses of heparin can interfere with platelet aggregation and thereby prolong bleeding time. It is unclear to what extent the antiplatelet effect of heparin contributes to the hemorrhagic complications of treatment with the drug. Heparin "clears" lipemic plasma in vivo by causing the release of lipoprotein lipase into the circulation. Lipoprotein lipase hydrolyzes triglycerides to glycerol and free fatty acids. The clearing of lipemic plasma may occur at concentrations of heparin below those necessary to produce an anticoagulant effect. Rebound hyperlipemia may occur after heparin administration is stopped.
Clinical Use. Heparin is used to initiate treatment of venous thrombosis and pulmonary embolism because of its rapid onset of action (Hirsh et al., 2001). An oral anticoagulant usually is started concurrently, and heparin is continued for at least 4 to 5 days to allow the oral anticoagulant to achieve its full therapeutic effect (see Clinical Use and Monitoring Anticoagulant Therapy). Patients who experience recurrent thromboembolism despite adequate oral anticoagulation (e.g., patients with Trousseau's syndrome) may benefit from long-term heparin administration. Heparin is used in the initial management of patients with unstable angina or acute myocardial infarction, during and after coronary angioplasty or stent placement, and during surgery requiring cardiopulmonary bypass. Heparin also is used to treat selected patients with disseminated intravascular coagulation. Low-dose heparin regimens are effective in preventing venous thromboembolism in certain high-risk patients. Specific recommendations for heparin use have been reviewed (Hirsh et al., 2001).
Low-molecular-weight heparin preparations were first approved for prevention of venous thromboembolism. They are also effective in the treatment of venous thrombosis, pulmonary embolism, and unstable angina (Hirsh et al., 2001). The principal advantage of low-molecular-weight heparin over standard heparin is a more predictable pharmacokinetic profile, which allows weight-adjusted subcutaneous administration without laboratory monitoring. Thus, therapy of many patients with acute venous thromboembolism can be provided in the outpatient setting. Other advantages of low-molecular-weight heparin include a lower incidence of heparin-induced thrombocytopenia and possibly lower risks of bleeding and osteopenia.
In contrast to warfarin, heparin does not cross the placenta and has not been associated with fetal malformations; therefore it is the drug of choice for anticoagulation during pregnancy. Heparin does not appear to increase the incidence of fetal mortality or prematurity. If possible, the drug should be discontinued 24 hours before delivery to minimize the risk of postpartum bleeding. The safety and efficacy of low-molecular-weight heparin use during pregnancy have not been adequately evaluated.
Absorption and Pharmacokinetics. Heparin is not absorbed through the gastrointestinal mucosa and therefore is given by continuous intravenous infusion or subcutaneous injection. Heparin has an immediate onset of action when given intravenously. In contrast, there is considerable variation in the bioavailability of heparin given subcutaneously, and the onset of action is delayed 1 to 2 hours; low-molecular-weight heparins are absorbed more uniformly.
The half-life of heparin in plasma depends on the dose administered. When doses of 100, 400, or 800 units/kg of heparin are injected intravenously, the half-lives of the anticoagulant activities are approximately 1, 2.5, and 5 hours, respectively (see Appendix II for pharmacokinetic data). Heparin appears to be cleared and degraded primarily by the reticuloendothelial system; a small amount of undegraded heparin also appears in the urine. The half-life of heparin may be shortened in patients with pulmonary embolism and prolonged in patients with hepatic cirrhosis or end-stage renal disease. Low-molecular-weight heparins have longer biological half-lives than do standard preparations of the drug.
Administration and Monitoring. Full-dose heparin therapy usually is administered by continuous intravenous infusion. Treatment of venous thromboembolism is initiated with a bolus injection of 5000 units, followed by 1200 to 1600 units per hour delivered by an infusion pump. Therapy routinely is monitored by the aPTT. The therapeutic range for standard heparin is considered to be that which is equivalent to a plasma heparin level of 0.3 to 0.7 units/ml as determined with an anti-factor Xa assay (Hirsh et al., 2001). The aPTT value that corresponds to this range varies depending on the reagent and instrument used to perform the assay. A clotting time of 1.8 to 2.5 times the normal mean aPTT value generally is assumed to be therapeutic; however, values in this range obtained with some aPTT assays may overestimate the amount of circulating heparin, and therefore be subtherapeutic. The risk of recurrence of thromboembolism is greater in patients who do not achieve a therapeutic level of anticoagulation within the first 24 hours. Initially, the aPTT should be measured and the infusion rate adjusted every 6 hours; dose adjustments may be aided by use of a nomogram (Hirsh et al., 2001). Once a steady dosage schedule has been established, daily monitoring is sufficient.
Very high doses of heparin are required to prevent coagulation during cardiopulmonary bypass. The aPTT is infinitely prolonged over the dosage range used. Another coagulation test, such as the activated clotting time, is employed to monitor therapy in this situation.
Subcutaneous administration of heparin can be used for the long-term management of patients in whom warfarin is contraindicated (e.g., during pregnancy). A total daily dose of about 35,000 units administered as divided doses every 8 to 12 hours usually is sufficient to achieve an aPTT of 1.5 times the control value (measured midway between doses). Monitoring generally is unnecessary once a steady dosage schedule is established.
Low-dose heparin therapy is used prophylactically to prevent deep venous thrombosis and thromboembolism in susceptible patients. (Until recently a suggested regimen for such treatment was 5000 units of heparin given every 8 to 12 hours.) The body of evidence now suggests that this regimen is clinically less effective than giving heparin every 8 hours in hospitalized medical and surgical patients at high risk for venous thromboembolism (Cade, 1982; Gardlund, 1996; Belch et al., 1981). Laboratory monitoring is unnecessary, since this regimen does not prolong the aPTT.
Low-Molecular-Weight Heparin Preparations. Enoxaparin (LOVENOX), dalteparin (FRAGMIN), tinzaparin (INNOHEP, others), ardeparin (NORMIFLO), nadroparin (FRAXIPARINE, others), and reviparin (CLIVARINE) differ considerably in composition, and it cannot be assumed that two preparations that have similar anti-factor Xa activity will produce equivalent antithrombotic effects. The more predictable pharmacokinetic properties of low-molecular-weight heparins, however, permit administration in a fixed or weight-adjusted dosage regimen once or twice daily by subcutaneous injection. Since they have a minimal effect on tests of clotting in vitro, monitoring is not done routinely. Patients with end-stage renal failure may require monitoring with an anti-factor Xa assay because this condition may prolong the half-life of low-molecular-weight heparin. Specific dosage recommendations for various low-molecular-weight heparins may be obtained from the manufacturer's literature. Nadroparin and reviparin are not currently available in the United States.
Synthetic Heparin Derivatives. Fondaparinux (ARIXTRA) is a synthetic pentasaccharide based on the structure of the antithrombin binding region of heparin. It mediates inhibition of factor Xa by antithrombin but does not cause thrombin inhibition due to its short polymer length. Fondaparinux is administered by subcutaneous injection, reaches peak plasma levels in 2 hours, and is excreted in the urine with a half-life of 17 to 21 hours. It should not be used in patients with renal failure. Because it does not interact significantly with blood cells or plasma proteins other than antithrombin, fondaparinux can be given once a day at a fixed dose without coagulation monitoring. Fondaparinux appears to be much less likely than heparin or low-molecular-weight heparin to trigger the syndrome of heparin-induced thrombocytopenia (see below). Fondaparinux is approved for thromboprophylaxis of patients undergoing hip or knee surgery (Buller et al., 2003) and for the therapy of pulmonary embolism and deep venous thrombosis. Idraparinux (undergoing phase III clinical testing as of 2004) is a more highly sulfated derivative of fondaparinux that has a half-life of 5 to 6 days; the lack of a suitable antidote may limit its clinical application.
Heparin Resistance. The dose of heparin required to produce a therapeutic aPTT varies due to differences in the concentrations of heparin-binding proteins in plasma, such as histidine-rich glycoprotein, vitronectin, and platelet factor 4; these proteins competitively inhibit binding of heparin to antithrombin. Occasionally a patient's aPTT will not be prolonged unless very high doses of heparin (>50,000 units per day) are administered. Such patients may have "therapeutic" concentrations of heparin in plasma at the usual dose when values are measured by other tests (e.g., anti-factor Xa activity or protamine sulfate titration). These patients may have very short aPTT values prior to treatment because of the presence of an increased concentration of factor VIII and may not be truly resistant to heparin. Other patients may require large doses of heparin because of accelerated clearance of the drug, as may occur with massive pulmonary embolism. Patients with inherited antithrombin deficiency ordinarily have 40% to 60% of the normal plasma concentration of this inhibitor and respond normally to intravenous heparin. However, acquired antithrombin deficiency (concentration less than 25% of normal) may occur in patients with hepatic cirrhosis, nephrotic syndrome, or disseminated intravascular coagulation; large doses of heparin may not prolong the aPTT in these individuals.
Toxicities. Bleeding. Bleeding is the primary untoward effect of heparin. Major bleeding occurs in 1% to 5% of patients treated with intravenous heparin for venous thromboembolism (Hirsh et al., 2001). The incidence of bleeding is somewhat less in patients treated with low-molecular-weight heparin for this indication. Although the number of bleeding episodes appears to increase with the total daily dose of heparin and with the degree of prolongation of the aPTT, these correlations are weak, and patients can bleed with aPTT values that are within the therapeutic range. Often an underlying cause for bleeding is present, such as recent surgery, trauma, peptic ulcer disease, or platelet dysfunction.
The anticoagulant effect of heparin disappears within hours of discontinuation of the drug. Mild bleeding due to heparin usually can be controlled without the administration of an antagonist. If life-threatening hemorrhage occurs, the effect of heparin can be reversed quickly by the slow intravenous infusion of protamine sulfate, a mixture of basic polypeptides isolated from salmon sperm. Protamine binds tightly to heparin and thereby neutralizes its anticoagulant effect. Protamine also interacts with platelets, fibrinogen, and other plasma proteins and may cause an anticoagulant effect of its own. Therefore, one should give the minimal amount of protamine required to neutralize the heparin present in the plasma. This amount is approximately 1 mg of protamine for every 100 units of heparin remaining in the patient; it is given intravenously at a slow rate (up to 50 mg over 10 minutes).
Protamine is used routinely to reverse the anticoagulant effect of heparin following cardiac surgery and other vascular procedures. Anaphylactic reactions occur in about 1% of patients with diabetes mellitus who have received protamine-containing insulin (NPH insulin or protamine zinc insulin) but are not limited to this group. A less common reaction consisting of pulmonary vasoconstriction, right ventricular dysfunction, systemic hypotension, and transient neutropenia also may occur after protamine administration.
Heparin-Induced Thrombocytopenia. Heparin-induced thrombocytopenia (platelet count <150,000/ml or a 50% decrease from the pretreatment value) occurs in about 0.5% of medical patients 5 to 10 days after initiation of therapy with standard heparin (Warkentin, 2003). The incidence of thrombocytopenia is lower with low-molecular-weight heparin. Thrombotic complications that can be life-threatening or lead to amputation occur in about one-half of the affected heparin-treated patients and may precede the onset of thrombocytopenia. The incidence of heparin-induced thrombocytopenia and thrombosis is higher in surgical patients. Venous thromboembolism occurs most commonly, but arterial thromboses causing limb ischemia, myocardial infarction, and stroke also occur. Bilateral adrenal hemorrhage, skin lesions at the site of subcutaneous heparin injection, and a variety of systemic reactions may accompany heparin-induced thrombocytopenia. The development of IgG antibodies against complexes of heparin with platelet factor 4 (or, rarely, other chemokines) appears to cause all of these reactions. These complexes activate platelets by binding to FcgIIa receptors, which results in platelet aggregation, release of more platelet factor 4, and thrombin generation. The antibodies also may trigger vascular injury by binding to platelet factor 4 attached to heparan sulfate on the endothelium.
Heparin should be discontinued immediately if unexplained thrombocytopenia or any of the clinical manifestations mentioned above occur 5 or more days after beginning heparin therapy, regardless of the dose or route of administration. The onset of heparin-induced thrombocytopenia may occur earlier in patients who have received heparin within the previous 3 to 4 months and have residual circulating antibodies. The diagnosis of heparin-induced thrombocytopenia can be confirmed by a heparin-dependent platelet activation assay or an assay for antibodies that react with heparin/platelet factor 4 complexes. Since thrombotic complications may occur after cessation of therapy, an alternative anticoagulant such as lepirudin, argatroban, or danaparoid (see below) should be administered to patients with heparin-induced thrombocytopenia. Low-molecular-weight heparins should be avoided, because these drugs often cross-react with standard heparin in heparin-dependent antibody assays. Warfarin may precipitate venous limb gangrene or multicentric skin necrosis in patients with heparin-induced thrombocytopenia and should not be used until the thrombocytopenia has resolved and the patient is adequately anticoagulated with another agent.
Other Toxicities. Abnormalities of hepatic function tests occur frequently in patients who are receiving heparin intravenously or subcutaneously. Mild elevations of the activities of hepatic transaminases in plasma occur without an increase in bilirubin levels or alkaline phosphatase activity. Osteoporosis resulting in spontaneous vertebral fractures can occur, albeit infrequently, in patients who have received full therapeutic doses of heparin (greater than 20,000 units per day) for extended periods of time (e.g., 3 to 6 months). Heparin can inhibit the synthesis of aldosterone by the adrenal glands and occasionally causes hyperkalemia, even when low doses are given. Allergic reactions to heparin (other than thrombocytopenia) are rare.
Other Parenteral Anticoagulants
Lepirudin. Lepirudin (REFLUDAN) is a recombinant derivative (Leu1-Thr2-63-desulfohirudin) of hirudin, a direct thrombin inhibitor present in the salivary glands of the medicinal leech. It is a 65-amino-acid polypeptide that binds tightly to both the catalytic site and the extended substrate recognition site (exosite I) of thrombin. Lepirudin is approved in the United States for treatment of patients with heparin-induced thrombocytopenia. It is administered intravenously at a dose adjusted to maintain the aPTT at 1.5 to 2.5 times the median of the laboratory's normal range for aPTT. The drug is excreted by the kidneys and has a half-life of about 1.3 hours. Lepirudin should be used cautiously in patients with renal failure, since it can accumulate and cause bleeding in these patients. Patients may develop antihirudin antibodies that occasionally cause a paradoxical increase in the aPTT; therefore, daily monitoring of the aPTT is recommended. There is no antidote for lepirudin.
Bivalirudin. Bivalirudin (ANGIOMAX) is a synthetic, 20-amino-acid polypeptide that directly inhibits thrombin by a mechanism similar to that of lepirudin. Bivalirudin contains the sequence Phe1-Pro2-Arg3-Pro4, which occupies the catalytic site of thrombin, followed by a polyglycine linker and a hirudin-like sequence that binds to exosite I. Thrombin slowly cleaves the Arg3-Pro4 peptide bond and thus regains activity. Bivalirudin is administered intravenously and is used as an alternative to heparin in patients undergoing coronary angioplasty. The half-life of bivalirudin in patients with normal renal function is 25 minutes; dosage reductions are recommended for patients with moderate or severe renal impairment.
Argatroban. Argatroban, a synthetic compound based on the structure of L-arginine, binds reversibly to the catalytic site of thrombin. It is administered intravenously and has an immediate onset of action. Its half-life is 40 to 50 minutes. Argatroban is metabolized by cytochrome P450 enzymes in the liver and is excreted in the bile; therefore dosage reduction is required for patients with hepatic insufficiency. The dosage is adjusted to maintain an aPTT of 1.5 to 3 times the baseline value. Argatroban can be used as an alternative to lepirudin for prophylaxis or treatment of patients with or at risk of developing heparin-induced thrombocytopenia.
Danaparoid. Danaparoid (ORGARAN) is a mixture of nonheparin glycosaminoglycans isolated from porcine intestinal mucosa (84% heparan sulfate, 12% dermatan sulfate, 4% chondroitin sulfate) with a mean mass of 5500 daltons. Danaparoid is approved in the United States for prophylaxis of deep venous thrombosis. It also is an effective anticoagulant for patients with heparin-induced thrombocytopenia and has a low rate of cross-reactivity with heparin in platelet-activation assays. Danaparoid mainly promotes inhibition of factor Xa by antithrombin, but it does not prolong the PT or aPTT at the recommended dosage. Danaparoid is administered subcutaneously at a fixed dose for prophylactic use and intravenously at a higher, weight-adjusted dose for full anticoagulation. Its half-life is about 24 hours. Patients with renal failure may require monitoring with an anti-factor Xa assay because of a prolonged half-life of the drug. No antidote is available. Danaparoid is no longer available in the United States.
Drotrecogin Alfa. Drotrecogin alfa (XIGRIS) is a recombinant form of human activated protein C that inhibits coagulation by proteolytic inactivation of factors Va and VIIIa. It also has antiinflammatory effects (Esmon, 2003). A 96-hour continuous infusion of drotrecogin alfa decreases mortality in adult patients who are at high risk for death from severe sepsis if given within 48 hours of the onset of organ dysfunction (e.g., shock, hypoxemia, oliguria). The major adverse effect is bleeding.
Philip W. Majerus and Douglas M. Tollefsen
key words: blood, Parenteral Anticoagulants, heparin
HEMOSTASIS: PLATELET FUNCTION, BLOOD COAGULATION, AND FIBRINOLYSIS
Hemostasis is the cessation of blood loss from a damaged vessel. Platelets first adhere to macromolecules in the subendothelial regions of the injured blood vessel; they then aggregate to form the primary hemostatic plug. Platelets stimulate local activation of plasma coagulation factors, leading to generation of a fibrin clot that reinforces the platelet aggregate. Later, as wound healing occurs, the platelet aggregate and fibrin clot are degraded.
Coagulation involves a series of zymogen activation reactions,. At each stage, a precursor protein, or zymogen, is converted to an active protease by cleavage of one or more peptide bonds in the precursor molecule. The components at each stage include a protease from the preceding stage, a zymogen, a nonenzymatic protein cofactor, Ca2+, and an organizing surface that is provided by a phospholipid emulsion in vitro or by platelets in vivo. The final protease generated is thrombin (factor IIa).
Conversion of Fibrinogen to Fibrin. Fibrinogen is a 330,000-dalton protein that consists of three pairs of polypeptide chains (designated Aa, Bb, and g) covalently linked by disulfide bonds. Thrombin converts fibrinogen to fibrin monomers by cleaving fibrinopeptides A (16 amino acid residues) and B (14 amino acid residues) from the amino-terminal ends of the Aa and Bb chains, respectively. Removal of the fibrinopeptides allows the fibrin monomers to form a gel, which is the end point of in vitro assays of coagulation (see below). Initially, the fibrin monomers are bound to each other noncovalently. Subsequently, factor XIIIa catalyzes an interchain transglutamination reaction that cross-links adjacent fibrin monomers to enhance the strength of the clot.
Structure of Coagulation Protease Zymogens. The protease zymogens involved in coagulation include factors II (prothrombin), VII, IX, X, XI, XII, and prekallikrein. About 200 amino acid residues at the carboxyl-terminal end of each zymogen are homologous to trypsin and contain the active site of the protease. In addition, 9 to 12 glutamate residues near the amino-terminal ends of factors II, VII, IX, and X are converted to g-carboxyglutamate (Gla) residues during biosynthesis in the liver. The Gla residues bind Ca2+ and are necessary for the coagulant activities of these proteins.
Nonenzymatic Protein Cofactors. Factors V and VIII are homologous 350,000-dalton proteins. Factor VIII circulates in plasma bound to von Willebrand factor, while factor V is present both freely in plasma and as a component of platelets. Thrombin cleaves V and VIII to yield activated factors (Va and VIIIa) that have at least 50 times the coagulant activity of the precursor forms. Factors Va and VIIIa have no intrinsic enzymatic activity but serve as cofactors that increase the proteolytic efficiency of Xa and IXa, respectively. Tissue factor (TF) is a nonenzymatic lipoprotein cofactor that greatly increases the proteolytic efficiency of VIIa. It is present on the surface of cells that do not normally contact plasma (e.g., macrophages and smooth muscle cells) and initiates coagulation outside a broken blood vessel. Monocytes and endothelial cells also may express tissue factor when exposed to a variety of stimuli, such as endotoxin, tumor necrosis factor, and interleukin-1. Thus these cells may be involved in thrombus formation under pathological circumstances. High-molecular-weight kininogen is a plasma protein that serves as the cofactor for XIIa when clotting is initiated in vitro in the activated partial thromboplastin time (aPTT) test.
Activation of Prothrombin. Factor Xa cleaves two peptide bonds in prothrombin to form thrombin. Activation of prothrombin by Xa is accelerated by Va, phospholipids, and Ca2+. When these components are all present, prothrombin is activated nearly 20,000 times faster than the rate achieved by Xa and Ca2+ alone. The maximal rate of activation occurs only when prothrombin and Xa both contain Gla residues, and therefore have the ability to bind to phospholipids. Purified platelets can substitute for phospholipids and Va to facilitate activation of prothrombin in vitro, provided that the platelets are stimulated to release endogenous platelet factor Va or that factor Va is added exogenously to unstimulated platelets. The surface of platelets that are aggregated at the site of hemostasis concentrates the factors required for prothrombin activation.
Initiation of Coagulation. Coagulation is initiated in vivo by the extrinsic pathway. Small amounts of factor VIIa in the plasma bind to subendothelial tissue factor following vascular injury. Tissue factor accelerates activation of factor X by VIIa, phospholipids, and Ca2+ about 30,000-fold. VIIa also can activate IX in the presence of tissue factor, providing a convergence between the extrinsic and intrinsic pathways.
Clotting by the intrinsic pathway is initiated in vitro when XII, prekallikrein, and high-molecular-weight kininogen interact with kaolin, glass, or another surface to generate small amounts of XIIa. Activation of XI to XIa and IX to IXa follows. IXa then activates X in a reaction that is accelerated by VIIIa, phospholipids, and Ca2+. Activation of factor X by IXa appears to occur by a mechanism similar to that for activation of prothrombin and may also be accelerated by platelets in vivo. Activation of factor XII is not required for hemostasis, since patients with deficiency of XII, prekallikrein, or high-molecular-weight kininogen do not bleed abnormally, even though their aPTT values are prolonged. Factor XI deficiency is associated with a variable and usually mild bleeding disorder. The mechanism for activation of factor XI in vivo is not known, although thrombin activates factor XI in vitro.
Fibrinolysis and Thrombolysis
The fibrinolytic system dissolves intravascular clots as a result of the action of plasmin, an enzyme that digests fibrin. Plasminogen, an inactive precursor, is converted to plasmin by cleavage of a single peptide bond. Plasmin is a relatively nonspecific protease; it digests fibrin clots and other plasma proteins, including several coagulation factors. Therapy with thrombolytic drugs tends to dissolve both pathological thrombi and fibrin deposits at sites of vascular injury. Therefore, the drugs are toxic, producing hemorrhage as a major side effect.
The fibrinolytic system is regulated such that unwanted fibrin thrombi are removed, while fibrin in wounds persists to maintain hemostasis (Lijnen and Collen, 2001). Tissue plasminogen activator (t-PA) is released from endothelial cells in response to various signals, including stasis produced by vascular occlusion. It is rapidly cleared from blood or inhibited by circulating inhibitors, plasminogen activator inhibitor-1 and plasminogen activator inhibitor-2, and thus exerts little effect on circulating plasminogen. t-PA binds to fibrin and converts plasminogen, which also binds to fibrin, to plasmin. Plasminogen and plasmin bind to fibrin at binding sites located near their amino termini that are rich in lysine residues (see below). These sites also are required for binding of plasmin to the inhibitor a2-antiplasmin. Therefore, fibrin-bound plasmin is protected from inhibition. Any plasmin that escapes this local milieu is rapidly inhibited. Some a2-antiplasmin is bound covalently to fibrin and thereby protects fibrin from premature lysis. When plasminogen activators are administered for thrombolytic therapy, massive fibrinolysis is initiated, and the inhibitory controls are overwhelmed.
Coagulation in Vitro. Blood clots in 4 to 8 minutes when placed in a glass tube. Clotting is prevented if a chelating agent such as ethylenediaminetetraacetic acid (EDTA) or citrate is added to bind Ca2+. Recalcified plasma clots in 2 to 4 minutes. The clotting time after recalcification is shortened to 26 to 33 seconds by the addition of negatively charged phospholipids and a particulate substance such as kaolin (aluminum silicate); this is termed the activated partial thromboplastin time (aPTT). Alternatively, recalcified plasma will clot in 12 to 14 seconds after addition of "thromboplastin" (a mixture of tissue factor and phospholipids); this is termed the prothrombin time (PT).
Two pathways of coagulation are recognized. An individual with a prolonged aPTT and a normal PT is considered to have a defect in the intrinsic coagulation pathway, because all of the components of the aPTT test (except kaolin) are intrinsic to the plasma. A patient with a prolonged PT and a normal aPTT has a defect in the extrinsic coagulation pathway, since thromboplastin is extrinsic to the plasma. Prolongation of both the aPTT and the PT suggests a defect in a common pathway.
Natural Anticoagulant Mechanisms. Platelet activation and coagulation normally do not occur within an intact blood vessel (Edelberg et al., 2001). Thrombosis is prevented by several regulatory mechanisms that require a normal vascular endothelium. Prostacyclin (prostaglandin I2; PGI2), a metabolite of arachidonic acid, is synthesized by endothelial cells and inhibits platelet aggregation and secretion. Antithrombin is a plasma protein that inhibits coagulation factors of the intrinsic and common pathways (see below). Heparan sulfate proteoglycans synthesized by endothelial cells stimulate the activity of antithrombin. Protein C is a plasma zymogen that is homologous to II, VII, IX, and X; its activity depends on the binding of Ca2+ to Gla residues within its amino-terminal domain. Activated protein C, in combination with its nonenzymatic Gla-containing cofactor (protein S), degrades cofactors Va and VIIIa and thereby greatly diminishes the rates of activation of prothrombin and factor X (Esmon, 2003). Protein C is activated by thrombin only in the presence of thrombomodulin, an integral membrane protein of endothelial cells. Like antithrombin, protein C appears to exert an anticoagulant effect in the vicinity of intact endothelial cells. Tissue factor pathway inhibitor (TFPI) is found in the lipoprotein fraction of plasma. When bound to factor Xa, TFPI inhibits factor Xa and the factor VIIa-tissue factor complex. By this mechanism, factor Xa may regulate its own production
Philip W. Majerus and Douglas M. Tollefsen
key words: blood, Prostacyclin, Anticoagulant Mechanisms, Fibrinolysis and Thrombolysis
THE COMPONENT OF THE BLOOD
The principal component of the blood plasma, which comprises over half of the total bood volume, is water. Our bodies are 70% water, our cells all function in a watery environment, and the blood is responsible for bringing adequate hydration to all of our tissues. The blood fluid also contains dissolved ions that are crucial for cell function. These ions are principally sodium, potassium, chloride, hydrogen, magnesium, and calcium. The plasma also transport iron, which is vital for crucial tissues enzymes but is particularly important for the structure and function of red blood cells.
The proteins of the blood plasma can be broadly divided into three classes: carrier proteins, immunoproteins, and coagulation proteins. The carrier functions of plasma proteins is threefold. First, the plasma proteins can bind molecules in the plasma, there by diminishing the nonspecific diffusion of these molecules into the tissues or their nonspecific interractions with blood and tissue cells. Second, complexes of carrier protein and molecules bound to them may be recognized by particular cells with a high degree to of specifity. In this way molecules such as iron can be directly targeted to cells that requaire them. Finally, plasma protein can diminish the toxic effect of certain molecules in the plasma by binding to them and by carrying these neutralize d toxins to specific sites, where they can be eliminate. Examples of these three carrier functions are evident for the plasma proteins describe in the following paragrahp.
The chief plasma protein is albumin. Comprising two-thirds of the mass of plasma protein, albumin is the major source of the osmotic pressure of thep plasma. In this sense albumin can be said to be a carrier of water, and when the serum albumin level declines the tissues of the body become edematous. Albuminis also a carrier of many compounds, examples of which are bilirubin and other bile pigments, and of free fatty acids. albumin doesn’t bind all such moleculeswith very high avidity, but because of the high albumin concentration, the proportion of these molecules that remain uncomplexed in the plasma is small. Another class of carrier proteins are the lipoproteins,which transport cholesterol, triglycerid, and phospholipid between tissues. Two plasma protein are particularly important for the transport of nutrients needed by the blood. One of these protein is transferrin, which carries iron to developing red blood cells. The second carrier is a class of proteins called transcobalamins, protein that transport cobalamins, vital cofactors for DNA synthesis in blood cells and other tissues.
Another set of carrier proteins complex to substances that may not ordinarily be present in the plasma but appear following injury or tissue dstruction. Haptoglobin is a protein that binds hemoglobin if this principal red blood cell connstituent appears in the plasma because of red blood cell destruction. A protein with a similar function is hemopexin, which bind to free heme released from denatured hemoglobin. Two proteins, alpha-1-antiprotease and alpha-2-macroglobulin, bind and neutralize proteolytic enzymes release from destroyed tissues and phagocytic leucocytes. The protease antiprotease-antiprotease complexes are avidly cleared from the circulation by mononuclear phagocytes. These antiprotease also function in the tissues. Injury and inflammation frequently increase the permeability of blood vesssels, allowing plasma constituent ti leak into the injured area. The antiproteases participate in the neutralization of proteolytic enzymes activated by the injury and inflammatory processes. An other ”anti-inflammatory” plasma protein is a copper binding protein called ceruloplasmin. Ceruloplasmin participates in the detoxification of oxygen free radicals that are released during inflammation by phagocytic cells. In addition, ceruplasmin is required to prevent the accumulation of copper in tissues, where it can have toxic effects. A conginetal disorder in which ceruloplasmin is absent, Wilson’s disease, is associated with extensive tissue damage, including red blood cell destruction.
Aside from nutrition, defense is the major function of the blood. Both cell and humors participate in this defense. The blood humors active in defense against invading micro-organism are the immunoproteins, the immunoglobulins, and the complemen proteins.
One of the tasks of the blood is to maintain the integrity of its conduit, the vascular system. As in defense against infection, cells and humors cooperate in this endeavor. The humoral arm of this defense is the coagulation system, a series of plasma proteins that interact to produce gelatinous plugs for sealing breaks and leaks in the vasculature. A variety of condition activate the coagulation protein. The solidificattion of plasma as a result of this activation is due to the formation of a network of strands composed of the protein fibrin. Between the strands of fibrin are water, salts, and the majority of plasma proteins. By centrifugation of the clot, these compound can be squeezed out; collectively they are called the serum. One of the stimuli for blood coagulation is contact with of the plasma with foreign surface such as glass or plastic. Therefore, any attempt to remove blood from the vascular system results in activation of the clotting system unless coagulation is inhibited in some way. For a number of diagnostic procedures involving blood and for blood blood transfusion it is inconvenient to have clotting occur. The coagulation system is very dependent on the presence of inonized calcium, so that if calcium is lowered by means of a suitable chelating agent coagulation is inhibited. Blood can be drawn into a container containing a sufficient quantity of sodium citrate or else ethylenediaminetetraasetic acid (commonly called EDTA) to complex all of the free calcium and will not clot. A system of proteins complementary to the coagulation proteins inhibits the coagulation system. It prevents the whole circulation from clotting up at once, if exposed locally to one of the many coagulation activators, and also break down formed clots when they are no longer needed. This system is called the fibrinolytic system.
key words: blood, tranobalamins, fibrinolytic system, Ceruloplasmin
(Bernard M. Babior & Thomas P. Stossel ; Hematology A Pathophysiological Approach, 1-3)
HEMATOLOGY
The discipline of hematology encompases the circulating blood and the function of blood cells throughout the body.Robert Burton, in 1628, characterized the blood as a “hot, temperate, red humor whose office is to nourish the whole body to give it strengthand color being dispersed by the veins through every part of it.” That elegantly simple and apt description of the bood can now be embellished by our deep understanding of the biology of this most excellent humor.
The mass of the circulating blood comprises 5 to 7 percent of our body weight. The blood is a suspension of cells, called a formed elements, in plasma, a solution of protein and salts. The bood and the circulatory system, the conduit of sluices, gates, and alleyways through which the blood courses, were required for the evolution of complex higher organisms the blood provides for nutritions, oxygenation, the cleansing of wastes, and the defense of body tissues against ever present asault by microbes.
Hematology is a subspecialty of internal medicine. Practitioners of this form of hematology are specialists in dealing with diseases of the bloodthey diagnose and treate anemias , coagulation disorders, and malignant neoplasms of blood cells. The treatment of malignancies of the blood is very similar to the treatment of a variety of solid tumors. Conversely, the teraphy of solid tumors often has profound effects on the blood.For these reasons, the disciplines of hematology and oncology have always been closely related., and in recent years have become even more closely linked. Another close relationship exist betweeen hematology and immunology. Abnormalities of the numbers and functions of white blood cells often cause a patient to become abnormally susceptible to infection, a state defined as immunodeficiency. On the other hand, perversion of the immune system so that it destroys normal blood cells, a state defined as autoimmunity, is frequently encountered in medical practice.
Another aspect of hematology is blood transfussion teraphy. Replacement of whole blood and blood components is a major medical enterprise, occuring during surgery and following injury. Not only are blood components infused into patients, but, increasingly, abnormal proteins are removed from patients with various diseases.
Finally, nearly every physician is, in a sense, A “Hematologist.” Because of its access to all tissues of the body, the blood is often one of the first sources of informations that indicate changes in the state of health and the development of specific diseases. Changes in the number or appearance of formed elements and in the levels of plasma components are extremely useful to all clinicians. It is not surprising, then, that diagnostic hematology laboratories are consulted by nearly all clinicians, and that the total impactof the work of these laboratories on the cost of health care is considrable.
(Bernard M. Babior & Thomas P. Stossel; 1984)
key words: Hematology, Hematologist, hematology and oncology, hematology and immunology
Monday, June 22, 2009
EFFECT OF BITTER MELON (Momordica charantia)
EFFECT OF BITTER MELON (Momordica charantia) LEAVES JUICE ON IMMUNOGLOBULIN M (IgM) AND IMMUNOGLOBULIN G (IgG) ACTIVITY OF MALE MICE (MUS MUSCULUS)
Arie Arizandi K., Hajar Astuti, Rangga Meidianto A., Mufidah
(Faculty of Pharmacy, Hasanuddin University, Makassar)
Abstract
Bitter melon (Momordica charantia) is widely growth in tropical area, and the leaves was useful in medicinal. Research about the effect of bitter melon leaves juice for immunomodulatory activity had been done. Three different concentrations, 25% w/v, 50% w/v, and 75% w/v of bitter melon leaves juice in water, with 1 ml/30 g body weight given orally once a day for six days. Then each animal was immunized 1 ml/ 30 g body weight of 2 % Sheep Red Blood Cells (RBC) as the antigenic challenges. Evaluation of Ig activity was conducted at the day 6th and 11th after immunization for IgM and IgG, respectively, using haemagglutinating antibody titter (HAT) method. The result of study indicated that juice of bitter melon leaves increase immunoglobulin M (IgM) four grade higher than negative control, while for the immunoglobulin G (IgG) are twice higher than negative control; and these improvement were statistically significant (P>0.01). The highest Ig activity was achieved by 25% w/v of bitter melon leaves juice.
Keywords : Bitter melon leaves, haemagglutinating antibody titter (HAT), immunoglobulin M (IgM), immunoglobulin G (IgG), sheep Red Blood Cells (RBC).
Introduction
Nowadays, one of the developed principal methods of medicinal that using traditional medicine is by increased the immunity system. If the disease include in infection disease, so the immunity system killed the causes of the disease by indirectly mechanism, that is increased the cellular defender. It is the reason of increasing of immunity system of the patient (Winarno et al.2000).
The defender of body is related with the presence of antibody. Actually, antibody is immunoglobulin protein that being secreted by B cell which is fixated by antigen. Immunoglobulin is the first substances that being identified as molecule in serum that can neutralized the amount of microorganism that caused infection disease. Immunoglobulin M (IgM) is the antibody that very efficient in complement activation, while immunoglobulin G (IgG) can help the phagocitocyt at the destruction of antigen (Bratawidjaja. 2004).
Momordica charantia is a tropical and subtropical vine of the family Cucurbitaceae, widely grown for edible fruit, which is among the most bitter of all vegetables. English names for the plant and its fruit include bitter melon or bitter gourd. (Abascal et al.,2005).
Bitter melons have been used in various Asian traditional medicine systems for a long time. Like most bitter-tasting foods, bitter melon stimulates digestion. While this can be helpful in people with sluggish digestion, dyspepsia, and constipation, it can sometimes make heartburn and ulcers worse. The fact that bitter melon is also a demulcent and at least mild inflammation modulator, however, means that it rarely does have these negative effects, based on clinical experience and traditional reports. (Abascal et al.,2005).
Also known as Ku gua, the herbaceous, tendril-bearing vine grows to 5 m. It bears simple, alternate leaves 4-12 cm across, with 3-7 deeply separated lobes. Each plant bears separate yellow male and female flowers. The young shoots and leaves may also be eaten as greens; in the Philippines, where bitter melon leaves are most commonly consumed, they are called dahon (leaves) ng ampalaya. The seeds can also be eaten, and give off a sweet taste, but have been known to cause vomiting and stomach upset. (Abascal et al.,2005).
Materials and Methods
Materials
Momordica charantia fresh leaves were collected in month of January from Makassar, Indonesia. The fresh leaves were diluted to made the juice with concentration 25% w/v, 50% w/v, and 75% w/v.
Animals
Male mice (20-30 g) were used, animals were housed under standard conditions of temperature (23°C±1°C). Fresh Sheep Red Blood Cell (SRBC) in alsevers solution was obtained from Makassar veterinary.
Antigen
Sheep Red Blood Cell (SRBC) collected in alsevers solution, washed three times in large volumes of pyrogen free 0.9% normal saline and adjusted to a concentration 2% for immunization and challenge.
Treatment
Bitter melon (Momordica charantia) leaves prepared in laboratory in a simple mortar using pestle; animals were divided into four group consisting mice served as control (Group I). the herbal formulation was fed orally at concentration 25% w/v/day (group 2), 50% w/v/day (group 3), and 75% w/v/day (group 4) for assessment immunomodulatory effect.
Haemagglutinating Antibody Titer (HAT)
Mice of group II until IV were pretreated with bitter melon leaves juice for six days and each mouse was immunized with 1ml/30g weight SRBC 2%/mouse by i.p. route. The day of immunization was referred as day 0 after treated with bitter melon leaves juice. After 5 days for IgM and 10 days for IgG, blood sample were collected from each mouse on day 6 for HAT. The titer was determined by tittering serum dilution with SRBC (0.05x109). The micro plates were incubated at 37oC for one hour and room temperature for 24 hours and examined visually for agglutination. The reciprocal of highest dilution of serum showing 50% agglutination has been expressed as HAT.
Statistical Analysis
The data were analyzed using Complete Random Device (CRD) method and then continued with Duncan’s Multiple Range Test (DMRT) method. p>0,05 were considered significant for both of treatment (IgM and IgG).
Results and Discussion
Results
Bitter melon (Momordica charantia) leaves juice was evaluated for Immunoglobulin M (IgM) and immunoglobulin G (IgG) activity. This showed a significant increased in Immunoglobulin M (IgM) and immunoglobulin G (IgG) activity (p>0,05) in mice.
The treatment induced marked enhancement of humoral response in animals. From the study it may be inferred that bitter melon (Momordica charantia) leaves juice promotes increased in Immunoglobulin M (IgM) and immunoglobulin G (IgG) activity and thus rationalizing its traditional claim.
Discussion
Immunomodulatory agents e.g IgM and IgG of the animal origin enhanced against a pathogen by activating the immune system. In the present study Momordica charantia when orally administrated, significantly increased the activity of IgM and IgG.
The data of immunoglobulin G (IgG) and immunoglobulin M (IgM) activity test after given the juice of bitter melon leaves depend on immunoglobulin titter towards to the mice after immunized of sheep Red Blood Cells (RBC) 2% are described on the table below :
Table the data of immunoglobulin M (IgM) activity.
| Treatment | Titer Immunoglobulin G (IgG) | |||
| Control | Cons. 25% | Cons. 50% | Cons. 75% | |
| IgM | 0.81 | 3.21 | 2.01 | 1.61 |
| IgG | 0.81 | 2.01 | 1.61 | 1.41 |
The activity of IgM in concentration 25% w/v was significantly increased when compared to untreated control.
The titer show little bit significant increased change with concentration 50% w/v and 75% w/v of bitter melon (Momordica charantia) leaves juice administration. However, a significant increase was observed at the concentration 25% w/v/day with 4 fold increase compared to control unrated animals (p>0,05). The augmentation of the humoral response as evidence by an enhancement of antibody responsiveness to SRBC in mice as consequently of pre and post immunization treatment indicates the enhanced responsiveness of b-lymphocytes subsets involve antibody synthesis. During Cell Mediated Immunity (CMI) response, sensitized T-lymphocytes, when challenged by the antigen, are converted to lymphoblast and secretes lymphokines, attracting more scavenger cells to the site of reaction. The infiltrating cells are thus immobilized to promote defensive. In our studies, foot volume was enhanced after bitter melon (Momordica charantia) leaves juice treatment suggests cell mediated enhancement (Sen et al.,1992).
Increase in HAT response indicated bitter melon (Momordica charantia) leaves juice potentiates humoral as well as cellular immunity. One of explanations for warded to justify the beneficial effects of indigenous drugs in diseases states is the non specific enhancement of immune states of the organism (Patil et al., 1998). The immunostimulant activity of bitter melon (Momordica charantia) leaves juice was known there was no documentary evidence.
Conclusion
Bitter melon (Momordica charantia) leaves juice has shown significant immunomodulatory effect in animals. Therefore clinical studies as a potential immunostimulat is further warranted. Therefore, we assume that clinical studies with bitter melon (Momordica charantia) leave juice in increased activity of immunoglobulin G (IgG) and immunoglobulin M (IgM) will result in positive outcome.
Acknowledgements
The authors are thankful to head, Faculty of Pharmacy, Hasanuddin University for providing the facilities and crew of department of immunological science, Medical Laboratory Makassar for helpful during this research.
References
Winarno, M. 2000. Penelitian Aktivitas Biologik Infus Benalu Teh (Scurulla atropurpurea Bl. Danser) terhadap aktivitas Sistem Imun Mencit. http://www.kalbefarma.com/files/06 Penelitian Aktivitas Biologik Infus Benalu Teh127.pdf/06 Penelitian Aktivitas Biologik Infus Benalu Teh127.html, diakses 24 Januari 2008.
Bratawidjaja, K. 2004.Imunologi Dasar. Edisi VI. Fakultas Kedokteran Universitas Indonesia. Jakarta. 19, 78, 82.
Patil,J.S., B.G., Nagavi, M. Ramesh and G.S. Vijayakumar. 1988. A study on the immunostimulant activity of Centella asiatica in rats.Indian Drugs., 35:711-714 .
Sen.P., P.K Mendiratta and A.Ray. 1992. Effect of Azardiracta indica on some biochemical, immunological and visceral parameters in normal and stressed rats. Ind. J. Exp. Biol., 30:1170-1175.
Abascal K, Yarnell E. 2005.Using bitter melon to treat diabetes. Altern Complemen Ther 11(4):179-184
Tuesday, June 16, 2009
Understanding ALL
Acute lymphocytic leukemia (ALL) is a type of blood cancer. Other names for ALL are acute lymphoblastic leukemia and acute lymphoid leukemia. About 5,430 people in the United States are expected to be diagnosed with ALL in 2008. It is the most common type of leukemia in children under age 15. The risk of getting ALL increases in people ages 45 and older. However, people can get ALL at any age. Most children with ALL are cured of their disease after treatment. ALL starts with a change to a single cell in the bone marrow. Scientists are studying the exact genetic changes that cause a normal cell to become an ALL cell. Few factors have been associated with an increased risk of developing ALL. Exposure to high doses of radiation therapy used to treat other types of cancer is one known risk factor. Other possible risk factors are continually under study. ALL is not contagious (catching). Information About Phosphocol P32. Phosphocol P32 is a prescription drug approved to treat adults with fluid in the abdominal or chest cavity caused by cancer or infection. Safety and effectiveness in children has not been established. The United States Food & Drug Administration (FDA) updated the safety information of this drug in August 2008 following reports linking Phosphocol P32 to leukemia, when used in an unapproved way to treat children with bleeding between the joints caused by hemophilia. The labeling of Phosphocol P 32 was modified by the manufacturer, Covidien Ltd. in August 2008 to reflect this risk. Information about the leukemia cases [two children (ages 9 and 14) with hemophilia developed acute lymphocytic leukemia approximately 10 months after intra-articular injections of Phosphocol P 32 (0.6 and 1.5 mCi total dose] was added to the "Warnings" section of the Phosphocol P 32 label. Also, "leukemia in children" is now noted as a risk in the label's "Adverse Events" section. Safety information is posted on the FDA Medwatch Web site (August 2008). The manufacturer strongly encourages medical professionals and their patients to follow the guidelines outlined in the prescribing information included with Phosphocol P 32. To read Covidien's communication to physicians click here August 29, 2008 manufacturer's letter to physicians. Some signs or symptoms of ALL are similar to other more common and less severe illnesses. Specific blood tests and bone marrow tests are needed to make a diagnosis. A person with ALL may have: The best advice for any person troubled by symptoms such as a lasting, low-grade fever, unexplained weight loss, tiredness or shortness of breath is to see a healthcare provider. Blood and bone marrow tests are done to look for leukemia cells. A CBC (complete blood count) is used to help diagnose ALL. A bone marrow aspirate and a bone marrow biopsy are two of the tests that are done. An aspirate is done to take a close look at the cells in the marrow in order to look for abnormal cells such as leukemic blast cells. It can also be used for cytogenetic analysis, immunophenotyping and other tests. The biopsy gives information about how much disease is in the marrow. Immunophenotyping is used to find out if the patient's leukemia cells are B cells or T cells. Most people with ALL have the B-cell type. Most cases of the B-cell type are called precursor B-cell type. The doctor uses information from these tests to decide the type of drug therapy a patient needs and how long treatment will last. Bone marrow tests are also done to see if treatment is destroying leukemic blast cells. To decide the best treatment for the patient, the doctor may also consider: Patients with ALL need to start chemotherapy right away. It is important to get medical care in a center where doctors are experienced in treating patients with ALL. The goal of treatment for ALL is to cure the disease. Children with ALL are likely to be cured of their disease. The number of adult patients who have remissions has increased. The length of remissions in adults has improved. There are two parts of treatment for ALL, called induction therapy and post-induction therapy. The aim of induction therapy is to: This is called a remission. Some drugs used to treat ALL are given by mouth. Other drugs are given by placing a catheter in a vein - usually in the patient's upper chest. During induction therapy most patients are treated with more than one drug and they may be given several drugs in combination. Each drug type works in a different way to kill the cells. Combining drug types can strengthen the effects of the drugs. Some of the drugs used to treat ALL are clofarabine, cytarabine, daunorubicin, methotrexate, mitoxantrone, cyclophosphamide, vincristine, pegaspargase, imatinib mesylate, prednisone and dexamethasone. Patients with ALL often have leukemic cells in the lining of the spinal cord and brain. The procedure used to check the spinal fluid for leukemic cells is called a spinal tap. The cells cannot always be found in an exam of the spinal fluid. To prevent leukemia in the central nervous system (CNS) leukemia, all patients who are in remission have the lining of the spinal cord and brain treated. In some cases, treatment is needed for ALL that has already affected the lining of the spinal cord and brain (CNS leukemia) and is causing problems such as headache, nausea and vomiting, and blurred vision. Parts of the body that aren't easily reached with chemotherapy given by mouth or IV - such as the lining of the spinal cord and brain - are treated by injection into the spinal fluid. Drugs such as methotrexate or cytarabine are injected into the spinal fluid either to prevent or treat CNS leukemia. When the treatment is for CNS leukemia, a spinal tap is done. Then spinal fluid is removed and chemotherapy is injected into the spinal canal. Radiation therapy may be given to the spine or brain. Spinal taps are done from time to time to check if leukemic cells are being killed and to give more doses of chemotherapy. Sometimes both chemotherapy and radiation therapy are used. Many ALL patients build up uric acid in their blood from their disease. Uric acid is a chemical made in the body. The use of chemotherapy also increases the uric acid. A high level of uric acid can cause kidney stones. Patients with high uric acid levels may be given a drug called allopurinol (Aloprim®, Zyloprim®) by mouth or IV. Another drug used to treat high uric acid levels is called rasburicase (Elitek®). Post-Induction Therapy High-risk types of ALL - such as T-cell ALL, infant ALL and adult ALL - are usually treated with higher doses of drugs during induction and post-induction therapy. One treatment plan is to use higher doses of drugs and give them for a longer time. Allogeneic stem cell transplant may be a good treatment for some high-risk ALL patients. About one out of five adults with ALL and a small number of children with ALL have a type called Ph-positive (or Philadelphia-positive) ALL. Ph-positive ALL may be treated with imatinib mesylate, also called Gleevec® or with other related drugs, such as dasatinib (Sprycel®) or nilotinib (Tasigna®). These drugs are given with chemotherapy. Gleevec® (or Sprycel® or Tasigna®) is given by mouth. Doctors are studying how well this treatment works in patients with Ph-positive ALL. During post-induction therapy, Gleevec® (or another related drug) is given with other drugs. Usually people with Ph-positive ALL stay on Gleevec® (or another related drug) after post-induction therapy is completed. Allogeneic Stem Cell Transplant Allogeneic stem cell transplant is a high-risk procedure. For this reason, it may not be a good treatment for some ALL patients. Allogeneic stem cell transplant may be a choice for adult ALL patients if: They are not doing well with other treatments. The expected benefits of stem cell transplant exceed the risks. There is a donor. Stem cell transplant is usually not considered for a child unless: Not all patients have treatment side effects. Patients who experience side effects should speak to their treatment teams about how to manage their side effects. Possible side effects of treatment for ALL include: To lower the risk of infection: Chemotherapy affects the parts of the body where new cells form quickly. This includes the inside of mouth and bowel, and the skin and hair. Some other chemotherapy side effects are: Drugs and other therapies can be given to prevent or treat nausea or vomiting. Follow-up Visits Treatment for ALL can cause long-term or late effects. Children should be checked for treatment effects on growth or learning that may not take place right away. It is important to identify problems early. Talk to the doctor about when your child's learning skills should be assessed. Some children will need special help with schoolwork during and after treatment. from : http://www.leukemia-lymphoma.org/all_page.adp?item_id=7049Causes and Risk Factors
ALL occurs at different rates in different geographic locations. There are higher rates in more developed countries and in higher socioeconomic groups. Scientists continue to explore possible relationships with life-style or environmental factors but no firm conclusions have yet been reached. This suggests that many factors may be involved. At the present time there is no known way to prevent most cases of the disease. Signs and Symptoms
Diagnosis
Treatment
More treatment is needed even after a patient with ALL is in remission. This is called post-induction therapy. It is given in cycles for two to three years. Post-induction therapy is given because some ALL cells remain that are not found by common blood or marrow tests. For most people, the postremission therapy drugs used are not the same drugs used during induction therapy. The doctor considers many things to decide the kind of post-induction therapy a patient needs, such as:Ph-Positive ALL-Induction/Post-Induction
Allogeneic stem cell transplant is a treatment used for some patients with ALL.
The main purpose of doing the transplant is to give strong doses of chemotherapy or radiation therapy to kill the ALL cells. This will also kill the healthy stem cells in the marrow. The transplanted donor stem cells help start a new supply of red cells, white cells and platelets.Side Effects of Treatment
Patients who have finished all of their therapy still need to go to their doctors regularly for exams and tests. The doctor may recommend longer periods of time between follow-up visits if a patient continues to be disease free.
