Journal of Thrombosis and Haemostasis, 1: 1471–1478

REVIEW ARTICLE

Heparin-induced thrombocytopenia B. H. CHONG Department of Medicine, St. George Clinical School, and Center of Thrombosis and Vascular Research, University of, New South Wales, Sydney, NSW, Australia

To cite this article: Chong BH. Heparin-induced thrombocytopenia. J Thromb Haemost 2003; 1: 1471–8.

Summary. Heparin-induced thrombocytopenia (HIT) is not only a common but also a potentially serious drug adverse effect. Unlike other drug-induced thrombocytopenias, HIT does not usually cause bleeding, but instead causes thrombosis. Thrombosis in HIT can lead to limb gangrene (requiring leg amputation) or even death. HIT is mediated by an antibody that recognizes an epitope on the platelet factor 4 (PF4)–heparin complex. The antibody–PF4–heparin complex binds to FcgRII receptors on the platelet surface and cross-links the receptors. This induces intense platelet activation and platelet aggregation, and simultaneously activates blood-coagulation pathways. These changes are probably the basis of the thrombotic events in HIT. Diagnosis of HIT should be made mainly on clinical criteria but should be confirmed whenever possible by laboratory tests, particularly in patients with comorbid conditions, in whom the diagnosis of HIT cannot be made with certainty without testing. The tests for HIT antibodies are either immunoassays (e.g. ELISA), or functional tests, (e.g. 14C-serotonin release assay). Once a clinical diagnosis of HIT is made, heparin should be ceased immediately and treatment with an alternative anticoagulant (such as danaparoid, r-hirudin or argatroban) commenced. This should continue for at least 5 days unless the diagnosis of HIT is subsequently proven to be incorrect. Warfarin should also be commenced when the patient is clinically stable and thrombosis is under control. There should be an overlap of a few days between warfarin and the alternative anticoagulant therapy. Keywords: antibody, heparin, platelets, thrombosis. Introduction Heparin is widely used for prevention and treatment of thrombotic disorders; however, it can cause serious adverse effects. One of these is heparin-induced thrombocytopenia (HIT), a common, serious and potentially life-threatening condition [1,2], and one for which diagnosis can be difficult [3]. Various Correspondence: Department of Medicine, WR Pitney Building, Level 2, St. George Hospital, Kogarah, NSW 2217, Australia. Tel.: þ 61 2 9350 2010; fax: þ 61 2 9350 3998; e-mail: beng.chong@unsw. edu.au # 2003 International Society on Thrombosis and Haemostasis

aspects of HIT will be discussed, including the frequency, pathophysiology, clinical features, diagnosis and management. Pathogenesis Types of HIT

Clinically there are two types of HIT, type I and type II [4,5]. In this review, when the term ‘HIT’ is used alone without specifying the type, it refers to type II. The mechanism of type I is still unknown but it is likely to be non-immune [4]. It is probably related to its platelet proaggregating effect. Heparin causes mild platelet aggregation in vitro [6,7], and possibly also causes mild platelet aggregation in vivo, particularly in the patients with activated platelets [8]. This results in increased platelet sequestration in the spleen and thrombocytopenia. Unlike type I, type II is mediated by an immune mechanism [4], and is caused by an antibody whose antigen is the heparin– platelet factor 4 (PF4) complex [9]. Formation of heparin–PF4 complex

PF4 is a highly positively charged tetrameric protein found in the a-granules of platelets. It is present in low levels in the plasma and its plasma concentration increases when PF4 is released from platelets following platelet activation. PF4 binds heparin very strongly and PF4–heparin complexes bind to platelets via the heparin binding sites [10]. When heparin binds to PF4, it induces a conformational change in the protein exposing certain kryptic epitope(s), to which the antibody binds [3]. Other negatively charged polysaccharides (heparan sulfate, polyvinylsulfonate, etc.) can also bind to PF4 and can induce a similar conformational change [11]. The binding of polysaccharides to PF4 is dependent mainly on their chain length and degree of sulfation [12]. For example, low molecular weight heparin (LMWH) is shorter in length than unfractionated heparin and binds weakly to PF4. It is less antigenic and causes HIT less frequently. The pentasaccharide is even shorter; it does not bind to PF4 and probably does not cause HIT. Formation of PF4–heparin complex occurs optimally at stoichiometric concentrations of PF4 and heparin. In the presence of high concentrations of heparin, the binding of the HIT

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antibody to the complex is disrupted. When heparin is administered as a continuous infusion, the plasma therapeutic drug concentration is 0.2–0.4 IU mL1 or 100–200 nmol L1 [13], and lower if it is administered subcutaneously for prevention of venous thromboembolism (VTE). In contrast to PF4, heparin is present in plasma in excess in most clinical situations. However, after an intravenous administration of heparin, PF4 is displaced from the surface of endothelial cells and the plasma PF4 level rises sharply to a maximum of about 8 nmol L1 [14]. This level is still below the stoichiometric concentration of PF4 that is optimal for the formation of heparin-PF4 complex. The optimal PF4 plasma concentration probably occurs in patients who have invivo platelet activation such as those undergoing hip surgery or cardiopulmonary bypass surgery. As expected, anti-PF4–heparin antibodies are frequently produced in these patients [15]. Binding of PF4–heparin–antibody complex to platelets

The HIT antibody binds heparin–PF4 complex with high affinity (Kd 13–30 nmol L1) [16]. The antibody–heparin–PF4 complex then binds to platelets via their Fc receptors (FcgRIIa) [17,18] (Fig. 1). Cross-linking of Fcg receptors on the platelet surface by the complex induces intracellular signaling and platelet activation, resulting in release of platelet granules and microparticles, thromboxane biosynthesis and ultimately platelet aggregation [19]. PF4, released from platelet a-granules during platelet activation, interacts with extracellular heparin to form more heparin–PF4 complexes, which bind to the surface of activated platelets. These cell surface-bound heparin–PF4 complexes serve as additional sites for HIT antibody binding [20]. Unlike the initial binding of heparin-PF4–antibody complexes to platelets, the subsequent binding of the antibody to PF4– heparin on activated platelets is via its Fab domain [20]. Two adjacent HIT antibody molecules bound to a platelet by their Fab domains still have their Fc domains free to interact, crosslink Fc receptors on a neighboring platelet, and induce platelet activation. Hence a chain of reactions are initiated, leading to intense platelet activation and platelet aggregation. Simultaneously, platelet procoagulant materials are generated; blood coagulation pathways are activated, resulting in thrombin gen-

eration and thrombus formation [21]. This prothrombotic process may be the basis for the hypercoagulable state and the frequent thrombotic complications seen in HIT [3,4]. On the other hand, the thrombocytopenia is largely due to the clearance of activated platelets and antibody-coated platelets by the reticulo-endothelial system. Transgenic mouse model of HIT

The mechanism of antibody–heparin–PF4–platelet interaction described in the previous section is based largely on in vitro data. Evidence supporting the mechanism also occurring in vivo came from a study by Reilly et al. [22]. Using a transgenic mouse model, these investigators demonstrated that human PF4 and FcgRIIa played a critical role in the pathogenesis of HIT and thrombosis. They showed that daily injections of 20 IU heparin daily and anti-hPF4–heparin antibody for five consecutive days to mice transgenic for human PF4 and human FcgRIIa resulted in severe thrombocytopenia. Under the same conditions, daily administration of 50 IU heparin led to widespread thrombosis and shock. In the control experiments, administration of heparin and a control antibody to hPF4/ hFcgRIIa transgenic mice had no effects. Furthermore, mice transgenic for hPF4 or hFcgRIIa alone did not develop thrombocytopenia or thrombosis when injected with heparin and antihPF4–heparin antibody. HIT antibody binding to endothelial cells

The HIT antibody reacts in vitro with PF4 on the endothelial cells [23,24] and causes immunoinjury to the cells. This in vitro reaction is independent of hFcgRIIa. However, whether this reaction occurs in vivo is uncertain. In hPF4 transgenic mice injected with heparin and anti-PF4/heparin antibody, the antibody should bind and induce damage to endothelial cells, and consequently cause thrombocytopenia and/or thrombosis, but neither occurred [22]. These data suggest that the HIT antibody may behave differently in vivo or that the antibody binding to endothelial cells may have no role in the pathogenesis of HIT.

Fig. 1. Interaction of the antibody, heparin and PF4 with platelets. The antibody reacts with heparin–PF4 complex to form antibody–antigen complexes, which then bind to the platelet via its Fcg receptors. Cross-linking the receptors leads to platelet activation and release of PF4 from the a granules. The released PF4 reacts with heparin to form heparin–PF4 complexes on the platelet surface, which serve as additional sites for HIT antibody binding. The free Fc domains of the attached antibodies can then cross-link Fc receptors of a neighboring platelet. # 2003 International Society on Thrombosis and Haemostasis

Heparin-induced thrombocytopenia 1473 HIT antibody: Ig subclasses

Most patients (>80%) have HIT IgG [4] antibodies, with or without IgA or IgM antibodies [25]. In some patients (50% without falling below normal platelet levels. The platelet counts do not return to normal unless heparin is stopped. Thereafter, the platelet count usually rises to above normal levels in 5– 7 days. This clinical picture is in sharp contrast with that of the patients with quinine-induced thrombocytopenia in whom the thrombocytopenia is severe (platelets < 10  109 L1) and its onset abrupt [3,44]. Thrombotic complications Thrombotic complications are common but bleeding is rare. The type and site of thrombosis varies according to the clinical setting [41,45]. For example, in postoperative patients who are prone to develop VTE, deep vein thrombosis (DVT) and pulmonary embolism occur very frequently [37]. The incidence of DVT in orthopedic patients who receive heparin for thromboprophylaxis is 20–30%, and the incidence increases dramatically to about 70% when these patients develop HIT [37]. Similarly, patients with central venous catheters and HIT develop upper limb venous thrombosis more frequently than those without HIT [46]. Venous thrombosis in patients with HIT is often extensive and severe, and can lead to venous gangrene or phlegmasia cerulea dolens, a condition rarely seen outside HIT [47]. Warfarin-induced protein C and protein S deficiency has been implicated as a significant etiological factor. In HIT patients with advanced atherosclerosis, arterial thrombosis in the lower limb is common and often results in limb gangrene and leg amputation [45]. Less frequently, arterial thrombosis results in stroke and acute myocardial infarction. Micro-vascular thrombosis (e.g. digital infarction) and disseminated intravascular thrombosis [48] also occur. Heparin resistance Heparin resistance occurs in some patients with HIT [49], due to high levels of circulating PF4 and other heparin-binding proteins, which are released into plasma from activated platelets. Acute systemic reactions Symptoms such as fever, tachycardia, flushing, headache, chest pain and dyspnea may occur in HIT patients following an intravenous bolus administration of heparin [51]. Acute amnesia, cardiac and pulmonary arrest have also been reported [52]. Diagnosis Clinical diagnosis

In any patient who develops thrombocytopenia while on heparin, HIT should always be considered. The diagnosis of HIT should be made firstly on clinical basis, based on the

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following criteria [1,2]: (i) thrombocytopenia occurs during heparin administration; (ii) other causes of thrombocytopenia have been excluded; and (iii) thrombocytopenia resolves after cessation of heparin. The term ‘thrombocytopenia’ is used loosely here. It refers to a drop of >50% in the patient’s platelet count from its baseline, as well as to a platelet decrease to below 100  109 L1 [3]. In Type II HIT, the decrease in platelets usually occurs between 5 and 10 days after commencement of heparin, but its onset can occur earlier if there has been prior exposure to heparin [44]. An onset after 10 days does not rule out the diagnosis. The onset of a new thrombosis or extension of a pre-existing thrombosis would further strengthen the clinical suspicion of HIT. Although criterion iii is not applicable at the onset of thrombocytopenia, it is helpful subsequently for confirmation of the diagnosis [53]. In the presence of comorbid conditions, such as other druginduced thrombocytopenias, infection, DIC and auto-immune thrombocytopenia the diagnosis of HIT is more difficult [3,53] as thrombocytopenia is also present in these conditions. To determine whether HIT coexists with one or more of these conditions, requires not only clinical judgement but also the results of laboratory tests for the anti-PF4/heparin antibody. Laboratory tests

Laboratory tests for HIT can be divided into two types [54]: functional tests and immunoassays. Details of the methodology of the tests are beyond the scope of this review. For this information, readers should consult other reviews or book chapters [33,54]. Functional tests Unlike other platelet antibodies, the HIT antibody causes strong platelet activation, which results in many platelet changes [4,19]. Some of these, such as serotonin release and platelet aggregation, have been used as the end-points in laboratory tests for HIT. The following functional tests have been described: (i) platelet aggregation test (PAT) [19]; (ii) 14Cserotonin release assay (SRA) [55]; (iii) heparin-induced platelet aggregation (HIPA) test [56]; (iv) ATP release using a lumiaggregometer [57]; (v) flow cytometry to detect platelet microparticle release [58]; (vi) annexin V binding to platelets [59]; and (vii) cell surface expression of P-selectin [60]. PAT, SRA and the HIPA test are commonly used but SRA is considered as the ‘gold standard’ [53,55]. The other tests are only used by a few laboratories. The sensitivity and specificity of the tests are influenced by a number of factors [33] including: 1 Use of platelet-rich plasma (PRP) or washed platelets. In general, tests using washed platelets are more sensitive because plasma contains high concentrations of IgG and other inhibitory proteins [61]. 2 Concentrations of heparin used for testing [33]. Optimal concentrations of heparin for a positive reaction are 0.1– 0.5 U mL1 [61]. High concentrations of heparin, e.g. 100 IU mL1, cause suppression of the reaction. This differential effect between the low and the high heparin concen-

tration is specific to the HIT antibody and has been exploited to increase the specificity of the test [55]. It is called the ‘twopoint system’ and it should be used, whenever possible, to obtain maximum specificity. 3 Variability of donors’ platelets [61,62]. Platelets used for testing vary from platelet donor to donor in their reactivity with the HIT antibody. It is important to use platelets from donors who are known to react well with the anti-PF4/heparin antibody so that maximum sensitivity can be obtained [61]. It is also important to use a weak HIT antiserum as a positive control to avoid a false negative reaction due to unreactive platelets. 4 Use of the anti-FcgRIIa antibody, IV.3 to increase specificity [3,18]. This has been recommended by some experts because IV.3 blocks platelet activation by the HIT antibody. IV.3 also inhibits platelet activation by other plasma stimuli, e.g. immune complexes and HLA allo-antibodies [61], which could give a false positive result. I find IV.3 to be unhelpful in this context and I do not recommend its use. In performing a functional assay to detect the HIT antibody, the operator should use the method he/she knows best but the factors discussed above must be taken into consideration so that maximum sensitivity and specificity can be achieved [33]. Immunoassays Four types of immunoassays for detection of the HIT antibody have been described. They are: (i) solid-phase antiPF4/heparin enzyme-linked immunosorbent assay (ELISA) [9]; (ii) PF4-polyvinylsulfonate antigen ELISA [11]; (iii) fluid-phase anti-PF4/heparin enzyme immunoassay (EIA) [63]; and (iv) particle gel immunoassay [64]. Solid-phase anti-PF4/heparin ELISA was first described by Amiral at al [9]. This assay is now available commercially, and detects the HIT antibody that binds to PF4/heparin complex in the microtiter well. We found a high background with this assay [63]. To circumvent this, we introduced a fluid-phase assay that has a low background. The fluid-phase assay [63] has not been widely taken up by others because it is tedious and timeconsuming. The PF4-polylsulfonate antigen assay is another solid-phase EIA that is commercially available [11]. In this assay, polylsulfonate, a highly negatively charged compound, replaces heparin in the target antigenic complex. This method has the advantage of the antigenic complex being stable for a longer period of time. Recently another commercial kit, particle gel immunoassay (Bio-Medical, Cressier sur Morat, Switzerland) [64] has been introduced. This method employs the particle gel technology widely used in red cell serology. Preliminary experience indicates that this semiquantitative assay is less sensitive than the other three immunoassays. Functional tests vs. immunoassay Compared with the functional assays, immunoassays are technically easier to perform and are also more sensitive [33]. This is confirmed by the findings of a recent ISTH serology survey. This survey also found that laboratories without the necessary experience and expertise did not perform the functional tests well, probably because they are technically demanding (B.H. Chong, unpublished data). However, # 2003 International Society on Thrombosis and Haemostasis

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the inexperienced laboratories do not have the same problem with the immunoassays. On the other hand, functional tests are better at detecting the HIT antibodies that are clinically significant and hence, if performed properly, they are more helpful in the diagnosis of HIT. Diagnosis: summary

The diagnosis of HIT is usually based on both clinical data and the results of laboratory tests. In the majority of patients, the clinical diagnosis is quite straightforward. Confirmation of the diagnosis by laboratory test results, though helpful, is less important. In some difficult cases, even after having taken into consideration all the available clinical data, the diagnosis is still uncertain. In these cases, the laboratory test results are particularly important in making the final decision as to whether or not the patients have HIT. Unfortunately, in many hospitals there is a delay before the test results are available. If so, the patient should be presumed to have HIT, and heparin should be stopped and if necessary, an alternative anticoagulant commenced without waiting for the test results. Management Type I

As the patient remains asymptomatic, no specific treatment is required [4]. It is sometimes difficult to differentiate Type I from Type II. In that situation it is safer to stop heparin and treat the patient as if they had Type II. Type II: with venous/arterial thrombosis

When Type II is suspected clinically, heparin should be withdrawn. When an acute venous or arterial thrombosis is present, an alternative anticoagulant should be commenced in its place [53]. The physician should use one of the three drugs that have been shown to be effective in HIT, danaparoid [65–67], lepirudin [68–70] or argatroban [71]. These drugs are immediately active and either inhibit thrombin directly or inhibit thrombin generation. Treatment with this drug should continue for at least 5 days or until the thrombosis is under control. To prevent recurrence of thrombosis, long-term treatment (e.g. for 6 months) with a vitamin K antagonist such as warfarin is usually required. Commencement of warfarin should be delayed a few days, particularly in the presence of severe or extending thrombosis, as protein C and protein S deficiency induced by warfarin can lead to thrombus progression and limb gangrene [47]. Treatment with danaparoid, lepirudin or argatroban should overlap warfarin therapy for a few days. Thrombolytic therapy (e.g. with streptokinase) may be lifeor limb-saving and is required in patients with severe DVT and impending limb gangrene, or in patients who have a massive pulmonary embolus with hemodynamic instability [72,73]. In some cases, embolectomy may be necessary [74]. Insertion of # 2003 International Society on Thrombosis and Haemostasis

an inferior vena caval filter is needed in patients who cannot be adequately anticoagulated. Danaparoid This is a mixture of heparan sulfate (84%), dermatan sulfate (12%) and other glycosaminoglycans (4%) [65]. Its anticoagulant effect is mediated via antithrombin III [75], and it has predominantly anti-FXa activity. It has a long plasma half-life of about 25 h and is excreted mainly in the kidneys [76]. In a prospective randomized study, danaparoid was shown to be more effective than dextran 70 in the treatment of HITassociated venous and arterial thrombosis [77]. In a compassionate use program, more than 460 patients with HIT-associated thrombosis were treated with danaparoid and there was a success rate of over 90% [78]. For treatment of HIT, patients are given an intravenous bolus dose of 2500 anti-Xa units followed by i.v. infusion of 400 U h1 for 4 h, then 300 U h1 for 4 h and subsequently 200 U h1 for another 5 days [65]. Although the intravenous regimen is preferred in the treatment of HIT, it can also be administered subcutaneously at a dose of 1500–2250 U b.i.d. [65]. In most cases of HIT, laboratory monitoring by measuring plasma anti-Xa levels is required. The dose of danaparoid should be adjusted to maintain plasma anti-Xa levels within 0.5–0.8 U mL1. Testing for cross-reactivity with danaparoid is unnecessary because in vitro cross-reactivity does not correlate with clinical response to treatment [63,79]. In vivo cross-reactivity occurs very rarely but when it occurs, danaparoid should be stopped and replaced by another anticoagulant [80]. r-Hirudin (Lepirudin) Hirudin is an anticoagulant protein, originally produced by the medicinal leech. It inhibits thrombin directly, both fluid phase and clot-bound [81]. It has a plasma half-life of 0.8–1.7 h after i.v. bolus injection of 0.1–0.5 mg kg1, and 1.1–2.0 h after continuous i.v. infusion [82]. It is also excreted mainly in the kidneys [83]. Two prospective, multicenter studies [68,69] (HAT-1, n ¼ 82; HAT-2, n ¼ 112) showed that HIT patients treated with lepirudin had more favorable clinical outcomes than historical controls (n ¼ 120). The combined endpoint (new thromboembolic complications, limb amputation and death) at day 35 was 52.1% in the historical control arm, while in the lepirudin arm, it was 25.1% in HAT-1 (P ¼ 0.024; adjusted risk ratio 0.5; 95% CI 0.29–0.89) and 31.9% in HAT-2 (P ¼ 0.15; adjusted risk ratio 0.7; 95% CI 0.44–1.14). In both studies, bleeding rate was higher in the lepirudin-treated patients (HAT-1, 39.1% and HAT-2, 44.6%) than in the control patients (27.2%). The dosing regimens for lepirudin are given below [82]:  Treatment for thromboembolism: 0.4 mg kg1 i.v. bolus followed by infusion of 0.15 mg kg1 h1  Treatment for thromboembolism (in patients who also received thrombolytic therapy): 0.2 mg kg1 i.v. bolus followed by infusion of 0.10 mg kg1 h1.  DVT prophylaxis in patients without thromboembolism: infusion of 0.1 mg kg1 h1. Laboratory monitoring with the activated partial thromboplastin time (APTT) is required. The APTT ratio should be kept

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in the therapeutic range of 1.5–2.5. In patients given lepirudin for >5 days, 45% develop IgG antihirudin antibodies [84]. The antibodies can either increase or decrease the APTT, and the lepirudin dose has to be adjusted accordingly. A few cases of anaphalaxis on re-exposure to the drug have been reported recently (A. Greinacher, personal communication). Argatroban This is a synthetic compound with molecular weight of 526 Da [85], which inhibits thrombin reversibly [86]. It is metabolized in the liver [87] and does not accumulate in the plasma of patients with renal failure. Its plasma half-life is short (40–50 min) [70]. Three prospective multicenter trials have been carried out [71]. Two were historical controlled studies and the third was a Phase III extension study. These studies showed that the incidence of the combined end-point (death, leg amputation and new thrombosis) was reduced in the argatroban-treated patients compared with that in the historical controls but the bleeding rate was similar. In these studies, argatroban was infused at 2 mg kg1 min1. The dose was regularly adjusted after 2 h and thereafter daily to keep the APTT between 1.5 and 3.0. Like danaparaoid and lepirudin, argatroban has no antidote. Type II: no clinically obvious thrombosis (isolated thrombocytopenia)

HIT is a hypercoagulable state [88]. In a retrospective study, about 53% of HIT patients who had only isolated thrombocytopenia initially, subsequently developed thrombosis, in all cases within 30 days [89]. These patients may benefit from treatment with one of the drugs discussed above [68–70]. Type II: special situations

Some patients with Type II HIT will need to undergo cardiac surgery. Danaparoid [33,65] and lepirudin [90,91] can be used instead of heparin during cardiopulmonary bypass in patients with HIT. Besides danaparoid [65,67] and lepirudin [92,93], argatroban [94] has also been used in patients with HIT during hemodialysis and hemofiltration. Due to space constraint, it is not possible to include a detailed discussion on the treatment of HIT in these situations. Similarly, treatment of HIT in pregnancy and childhood will not be discussed. Readers are referred to other reviews [33,65,82] for this information. References 1 Chong BH. Heparin induced thrombocytopenia. Br J Haematol 1995; 89: 431–9. 2 Chong BH. Heparin induced thrombocytopenia. Blood Rev 1988; 2: 108–14. 3 Warketin TE, Chong BH, Greinacher A. Heparin induced thrombocytopenia: towards consensus. Thromb Haemostas 1998; 79: 1–7. 4 Chong BH, Pitney WR, Castaldi PA. Heparin–induced thrombocytopenia: association of thrombotic complication with a heparin-dependent igg antibody which induced platelet aggregation, release and thromboxane synthesis. Lancet 1982; ii: 1246–8.

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