Pathophysiology of Heparin-Induced Thrombocytopenia.
Among the known side effects of heparin therapy, thrombocytopenia is without doubt the most frequent and dangerous. There are 2 types of heparin-induced thrombocytopenia (HIT). Heparin-induced thrombocytopenia I is characterized by a transitory and asymptomatic reduction in the platelet count, rarely below 100 x [10.sup.9]/L, that resolves spontaneously and does not require removal of the drug. The origin of HIT I is not yet completely known, but is thought to be related to a phenomenon of heparin-induced platelet clumping.[1-3] No immunologic components are involved in HIT I, and pathologic manifestations are rare.
Heparin-induced thrombocytopenia II has an immunologic origin[4,5] and is characterized by a significant reduction in platelets ([is greater than] 30%), generally after the fifth day of therapy; in the case of previous exposure to heparin, thrombocytopenia may appear earlier. The thrombocytopenia usually resolves 5 to 15 days after heparin has been removed, but in some cases it may take months.[6-8] The pathophysiologic manifestations of HIT II are complex and involve thrombosis at arterial, venous, and microvascular sites.
The true incidence of HIT II is not well defined because reported studies are mostly retrospective and differ regarding the characteristics of the patients considered, type of heparin administered, dosage, route of administration, duration of therapy, definition of thrombocytopenia, and laboratory tests employed for diagnostic confirmation (Table 1).[7,9,10] With reference to prospective studies in which the diagnosis was clinically based, there are important differences in the definition of thrombocytopenia. In some studies, the threshold value is 150 x [10.sup.9]/L,[11-17] while in others it is 100 x [10.sup.9]/L[18-30]; other investigators use the percent decrease as their reference. A relationship between the incidence of HIT II (defined only on a clinical basis), dosage, and type of unfractionated heparin used emerged from a study by Warkentin. The incidence was about 5% for therapeutic dosages of bovine heparin and 1% for porcine heparin, while it was less than 1% with prophylactic dosages of porcine heparin. In this series, the incidence of secondary thrombotic complications was about 20%. In a later review of prospective clinical trials, the incidence of HIT II varied from 1% to 30% in patients treated with high dosages of intravenous heparin, while it was less than 2% in patients administered low dosages of subcutaneous heparin.
Table 1. Epidemiology of Heparin-Induced Thrombocytopenia (HIT) General consensus of incidence <5% due to treatment with bovine heparin 1% due to treatment with porcine heparin <1% due to prophylaxis Thrombotic complications in 20% to 30% of HIT patients Incidence numbers depend on Patient characteristics Type of heparin Dose Route of administration Duration of therapy Definition of thrombocytopenia Confirmation by laboratory test
Schmitt and Adelman reviewed 23 randomized or cohort prospective studies for a total of 2160 patients in order to evaluate the impact of the various methodologic characteristics, such as HIT II definition, frequency with which platelet count was verified, and diagnostic criteria. This analysis confirmed that the incidence of HIT is overestimated in studies that do not include a "repeatedly abnormal platelet count." The cumulative incidence of HIT II in studies that employed "a reproducibly lowered platelet count" was 2.9% for bovine heparin and 1% for porcine heparin, and 1.7% for intravenous administration and 0% for subcutaneous administration. Even if this trend does not reach statistical significance, it speaks in favor of porcine heparin and subcutaneous administration of low dosages.
Our retrospective study disclosed a higher incidence. Independent of the route of administration, 6% of the patients had a clinical score suggestive of HIT II, with a 30% incidence of thrombotic complications and a 30% mortality rate; these values are in line with other published reports.[6,8,34] However, using more selective clinical criteria, the percent incidence lowered to 3% and the diagnosis was confirmed by the presence of anti-heparin-platelet factor 4 (anti-H-PF4) antibodies in only a fraction of patients.
On the other hand, Kappers-Klunne et al reported a particularly low (0.3%) HIT incidence in 558 cardiologic and neurologic patients treated with intravenous heparin. In this study, both functional and immunologic tests were used for laboratory confirmation of the clinical diagnosis. Anecdotal reports[36,37] describe HIT II induced by low-molecular-weight heparin (LMWH), but one clinical study indicated that its use is associated with a lesser incidence of thrombocytopenic and thrombotic complications than heparin.
A recent double-blind randomized study compared subcutaneous heparin with LMWH in 655 patients undergoing orthopedic surgery; the clinical diagnosis of HIT II was confirmed by means of the radioactive carbon ([sup.14]C)-serotonin-release assay (SRA). Heparin-induced thrombocytopenia II was documented in 2.7% of the patients treated with subcutaneous heparin and in none of the patients receiving LMWH (P = .0018). Thrombotic complications were also more frequent in the former (88.9%) than in the latter (17.8%) group (P [is less than] .001). In a subgroup of patients, independent of the presence of HIT II, more heparin-treated than LMWH-treated patients had a positive functional test (7.8% vs 2.2%, P = .02); thrombotic episodes, however, were more frequent in the patients who developed HIT II than in those with only a positive functional test. Thus, the frequency of laboratory-confirmed HIT II seems to be about 2% in patients receiving heparin, while it is much lower in those who receive LMWHs.
An immunologic basis of HIT II was first advocated by Rhodes et al, who showed that the immunoglobulin (Ig) G fraction from the serum of patients with HIT caused in vitro platelet aggregation in the presence of therapeutic concentrations of heparin. It has been reported that immunoglobulin-heparin complexes form only in the presence of platelets (Table 2). The proaggregating effect of heparin depends on the degree of sulfation and the molecular weight,[41-43] and is mediated by the release of substances from platelet [Alpha]-granules. Several platelet proteins/chemokines were proposed as the putative receptors of heparin-dependent antibodies, and PF4 was identified as the main cofactor.[46,47] The ratio of heparin to PF4 is critical for the constitution of the multimolecular antigenic complex, with an optimal heparin-PF4 ratio ranging from 1:4 to 1:6.[6,43,48-50] The antibody is not exclusively specific for the heparin-PF4 (H-PF4) complex, but also marks complexes of PF4 and other glycosaminoglycans.[41-43,51]
Table 2. Pathophysiology of Heparin-Induced Thrombocytopenia(*)
Current knowledge Antibody to heparin-PF4 complex Antibody to other chemokines and glycosaminoglycans IgG, also IgA and IgM Antibody binds to platelet Fc receptor and/or direct binding to platelet Functional heterogeneity of antibodies Up-regulation of cell and adhesion molecules/inflammatory response Preactivation of platelets/endothelial cells/white blood cells? Predisposing factors for thrombosis
(*) Ig indicates immunoglobulin.
The Figure shows a diagrammatic representation of a modified version of the currently accepted mechanism of action and pathophysiology of HIT (Table 2). At therapeutic concentrations ranging from 0.1 to 1.0 U/mL, heparin displaces PF4 from endothelial heparan sulfate or releases it directly from the platelets. Numerous PF4 molecules bind to heparin components, and the complex becomes immunogenic. As illustrated in the Figure, the immune complexes made up of anti-H-PF4 antibodies leading to the generation of 3 groups of antibodies (mainly IgG) activate the platelets and provoke an immune-mediated endothelial lesion[4,6,47,51,52] with thrombocytopenia, thrombosis, or both. The IgG anti-H-PF4 immune complex activates the platelets through the bond with the Fc[Gamma]RIIa (CD32) receptor, whose platelet surface expression ranges from 700 to 4000 binding sites and is further increased by the immune complex bond.[54,55] Platelet activation is blocked by both the monoclonal antibody (IV.3) specific for the Fc[Gamma]RIIa receptor[50,56] and the [F(ab').sub.2] fractions from patients with HIT II.[56,57] The Arg/His polymorphism at position 131 of the Fc[Gamma]RIIa receptor influences platelet reactivity to the immune complexes; in particular, the His/ His phenotype is more reactive to the Ig[G.sub.2] isotype. Nonetheless, while some studies have demonstrated a greater prevalence of HIT II and thrombotic complications in subjects with the His/His phenotype, others have not confirmed these findings. Other data are consistent with the hypothesis that H-PF4 complexes bind directly to platelets, and these complexes are the target for the [F(ab').sub.2] fraction of the antibody.
[Figure ILLUSTRATION OMITTED]
How the anti-H-PF4 antibodies cause thrombosis is not clear In general, Ig[G.sub.2]-isotype antiheparin antibodies are not particularly more frequent than the other subclasses in patients with HIT II, and IgM and IgA, which are not able to bind to Fc[Gamma]RIIa receptors, are also present in a significant percentage of these patients.[62-65] This finding suggests that the mechanism of platelet activation may occur independent of the Fc[Gamma]RIIa receptor for IgG. Moreover, the antibody isotype tends to modify in relation to the duration of the treatment.[49,61-64] The antibodies are still detectable in patient serum for about 4 to 6 weeks, and cases of antibody persistence for longer periods of time have been described.[6,33] Although the thrombotic complications in HIT syndrome are well described, only limited data have become available on the inflammatory components in this disease.
We have proposed a functional heterogeneity of anti-H-PF4 antibodies, based on the fact that heparin is a heterogeneous mixture of sulfated mucopolysaccharides with molecular, structural, and physical heterogeneity. Thus, heparins likely form multiple complexes with PF4, and depending on the nature of this interaction, the allosteric modifications in PF4 leading to a neoantigen formation may also vary. To characterize the anti-H-PF4 antibody in terms of functional heterogeneity, we obtained IgG fractions from the serum of patients with HIT II utilizing ammonium sulfate precipitation (ASP) and H-PF4-sepharose 4B affinity chromatography methods. With affinity purification, 2 major components, peaks 1 and 2, with high anti-H-PF4 antibody titers were eluted (purity was established by sodium dodecyl sulfate-polyacrylamide gel electrophoresis). While peak 1 (despite having a high anti-H-PF4 antibody titer) did not induce serotonin release from platelets in a heparin-dependent manner, peak 2 and the IgGs obtained with the ASP method exhibited a strong and concentration-dependent activation in the presence or absence of heparin (as well as LMWHs) (Table 3). These data suggest the generation of "superactive" HIT antibodies capable of activating platelets without heparin. The anti-H-PF4 antibody titers of peak 1, peak 2, and the ASP-IgG as measured by the heparin-induced platelet aggregation test-enzyme-linked immunosorbent assay (ELISA) (Stago, Asnieres, France) were similar.
Table 3. Platelet Activation in the Presence of 0.1 U/mL Heparin and Heparin-Induced Thrombocytopenia (HIT) Immunoglobulin Preparations(*)
% Serotonin Release Sample Range Mean [+ or -] SD HIT-negative serum 0-5 3.2 [+ or -] 1.1 HIT-positive serum 60-92 76 [+ or -] 6.2 Ig peak 1([dagger]) 2-8 4.1 [+ or -] 3.2 Ig peak 2([dagger]) 70-95 79 [+ or -] 3.6 IgG-ASP([double dagger]) 35-92 63 [+ or -] 9.7
(*) Ig indicates immunoglobulin; ASP, ammonium sulphate precipitation.
([dagger]) Isolated from patients (n = 6) clinically diagnosed with HIT II; protein content adjusted to 5 [micro]g/mL.
([double dagger]) Isolated from plasma samples of patients (n = 6) clinically diagnosed with HIT II; protein content adjusted to 50 [micro]g/mL.
To rule out the possible presence of a heparin contamination in these IgG preparations (and thus to confirm antibody activity independent of heparin), heparinase digestion, the use of an ion-exchange resin (Heparsorb), and dialysis against 1x phosphate-buffered saline were performed on the HIT sera, peak 2, and IgG-ASP. None of these treatments resulted in a significant decrease in the activation of platelets. These observations underscore the complex pathophysiology of HIT syndrome and suggest that there may be an HIT antibody active in a non-heparin-dependent manner.
Because the pathophysiology of HIT II involves the activation of platelets, endothelial cells, and leukocytes, it is reasonable to assume that cellular activation products, such as soluble selectins, cellular adhesion molecules, or both, would be increased in HIT. Our studies showed that selectin levels were markedly elevated in HIT II patients (Table 4). Treatment of these patients with the direct thrombin inhibitor argatroban (Novastan, Texas Biotechnology Corp, Houston, Tex) was associated with some decrease in the level of selectins at 24 and 72 hours posttreatment. A similar decrease of soluble P-selectin levels was also noted when HIT patients were treated with other direct thrombin agents (eg, hirudin and hirulog) as alternative anticoagulation therapies. Since thrombin plays an important role in the activation of platelets, resulting in microparticle formation, the selectin-level down-regulation may be related to the inhibition of thrombin generation. In addition, we have observed an increase in the intracellular adhesion molecules and the vascular cell adhesion molecules in HIT patients.
Table 4. Soluble P-, E-, and L-Selectin Levels in Patients From the ARG 911 Clinical Trial of Heparin-Induced Thrombocytopenia(*)
Normal Marker (n = 20) sP-Selectin, ng/mL 27 [+ or -] 8 sE-Selectin, ng/mL 63 [+ or -] 12 sL-Selectin, ng/mL 1245 [+ or -] 140 Pretreatment Marker (n = 30-40) sP-Selectin, ng/mL 98 [+ or -] 10([dagger]) sE-Selectin, ng/mL 101 [+ or -] 15([double dagger]) sL-Selectin, ng/mL 1690 [+ or -] 40([double dagger]) 24 h Posttreatment Marker (n = 30-40) sP-Selectin, ng/mL 78 [+ or -] 7([dagger]) sE-Selectin, ng/mL 95 [+ or -] 14([double dagger]) sL-Selectin, ng/mL 1525 [+ or -] 165([sections]) 72 h Posttreatment Marker (n = 30-40) sP-Selectin, ng/mL 69 [+ or -] 6([dagger]) sE-Selectin, ng/mL 80 [+ or -] 11([double dagger]) sL-Selectin, ng/mL 1406 [+ or -] 149([sections])
(*) Citrated blood plasma samples were collected at pretreatment and after 24 and 72 hours of argatroban anticoagulation (2-10 [micro]g/kg/min infusion adjusted to a therapeutic activated partial thromboplastin time of 60-100 seconds). The selectins were measured using ELISA-based assays (R&D Systems). NS indicates not significant.
([dagger]) P < .01 compared to the normal control.
([double dagger]) P < .05 compared to the normal control.
([sections]) Not significant.
In an experimental mouse model, the formation of autoantibodies against the H-PF4 complex produced thrombocytopenia but not thrombosis. From a pathogenic point of view, it is likely that a state of platelet and endothelial cell preactivation and probably other unidentified factors contribute to the thrombotic phenomena.[6,52,69,70]
In HIT II, the onset of thrombocytopenia appears to be independent of the type of heparin, dosage, and route of administration. The entity of thrombocytopenia usually varies from 50 to 100 x [10.sup.9]/L, but severe cases are frequent (Table 5).[7,71] There is no gender predominance, although elderly patients undergoing postsurgical prophylaxis or treatment for deep vein thrombosis,[71,72] in particular orthopedic and cardiovascular surgery, seem to be at higher risk. In more than 60% of the cases, other concomitant prothrombotic factors exist, such as diabetes, neoplasm, cardiac insufficiency, systemic lupus erythematosus, antiphospholipid syndrome, infection, and trauma. Besides thrombocytopenia, cutaneous allergic manifestations and skin necrosis may be present.
Table 5. Clinical Presentation of Patients With Heparin-Induced Thrombocytopenia
Typically 50-100 x [10.sup.9]/L or less; no apparent thrombocytopenia observed in some patients No gender predominance Higher risk of thrombosis in elderly, following orthopedic surgery, and following cardiovascular surgery Other concomitant prothrombotic factors Skin necrosis/cutaneous allergic manifestation Arterial and venous thrombosis
Despite the thrombocytopenia, hemorrhagic events are not frequent, while the major clinical complication is thrombosis. Both arterial and venous thrombosis can complicate the course of HIT II. A worsening of the thrombosis or a new thromboembolic complication necessitates the initial heparin treatment.[8,9,32,73] The thrombotic complications may appear even in the absence of thrombocytopenia. Arterial thrombosis was the first reported event to be associated with HIT[39,75]; nonetheless, today arterial and venous thrombotic complications are commonly found in HIT patients. Arterial thrombosis seems to be more frequent in patients with cardiovascular disease,[34,71] and venous complications are found more often in patients undergoing postsurgical prophylaxis.[8,9,34,71] The most common arterial complications are thromboses of the large vessels with gangrene and limb amputation, stroke, myocardial infarction, and cardiac thrombosis.[4,5,32,34,71] Venous complications are deep vein thrombosis, pulmonary embolism, thrombosis of the cerebral venous sinus, and closure of arterial-venous fistula in dialyzed patients; disseminated intravascular coagulation and hemorrhagic adrenal necrosis have been documented occasionally.[4-7,32]
It is commonly believed that HIT II is underdiagnosed. During heparin therapy, platelet counts must be checked regularly, at least twice weekly, especially in patients receiving treatment for more than 4 days, in those who show resistance to heparin, or in patients who have treatment-related skin manifestations (Table 6). Once thrombocytopenia is confirmed, the diagnosis of HIT II should be formulated on the basis of clinical criteria and the in vitro demonstration of heparin-dependent antiplatelet antibodies.[5,7] Nonetheless, the diagnosis is still only clinically based (associated with negative laboratory results) in more than 20% of cases. To evaluate the clinical probability of HIT II, various scoring systems have been proposed based on the presence of thrombocytopenia, recovery following drug suspension, onset of thrombotic or cutaneous complications, and the exclusion of other causes of thrombocytopenia.[77,78] A score less than 3 is not associated with a diagnosis of HIT II. Heparin-induced thrombocytopenia II is possible with a score from 4 to 6, and a score greater than 6 is highly probable for HIT II.
Table 6. Diagnosis of Heparin-Induced Thrombocytopenia (HIT)
Platelet counts twice weekly, especially in patients with Heparin treatment > 4 d Heparin resistance Related skin manifestations Clinical score system(*) Thrombocytopenia Recovery following heparin discontinuation Thrombosis Cutaneous complications Exclusion of other causes Laboratory confirmation [sup.14]C-Serotonin release assay (platelet function test) HIT serum/heparin-induced platelet aggregation (platelet function test) Antibody titer to heparin-platelet factor 4 ELISA (immunologic test)
(*) Score <3 indicates not HIT; 4-6, possible HIT; >6, highly probable HIT.
Among the functional laboratory tests, the SRA is the reference procedure. This test is based on the capacity of heparin-dependent HIT antibodies to induce the release of [sup.14]C-serotonin from platelets. Serum from a patient suspected of having HIT II is incubated with therapeutic heparin concentrations (0.1-1.0 U/mL) and washed donor platelets labeled with [sup.14]C-serotonin. If heparin-dependent antibodies are present, platelets are activated and the labeled serotonin is released. In the presence of high heparin concentrations (10-100 U/mL) the release is inhibited. This method has several disadvantages in that it requires the use of radioactive material, the result is highly dependent on the characteristics or reactivity of the donor platelets,[79,80] and it is time-consuming.
The platelet aggregation test, which utilizes a principle similar to that of the SRA, is able to furnish quicker results. This test measures platelet aggregation induced by the HIT patient serum in the presence of therapeutic concentrations of heparin. While this method is widely used due to its relative simplicity, the results vary considerably more than those reported for SRA in relation to the different heparin concentrations and donor platelet variability.[5,7,32,80,81] The overall sensitivity of this test is less than that of the SRA.[80,81]
The heparin-induced platelet aggregation test on micro-ELISA plates demonstrates greater reliability and correlation with the SRA than the platelet aggregation test. This test is based on the visual evaluation of the aggregation of washed platelets from different donors in the presence of heparin utilizing a magnetically shaken microplate.
Regardless of the functional method used to detect HIT antibodies, the selection of the donor platelets is crucial.[79,80] Under the most optimal conditions, the sensitivity of the aggregation and the SRA methods can reach 88% and 94%, respectively.[4,5,7,32,83] The typical response, however, is 50% to 60% sensitivity of these assays.
Recently, other functional tests have been suggested, such as the bioluminescent adenosine nucleotide-release assay or the binding of annexin V to platelet membrane anionic phospholipids utilizing flow cytometry. As these tests also employ donor platelets, they too would be affected by platelet variability. Other flow cytometric assays have been developed using small volumes of patient platelets or whole blood.[85,86] This approach could provide a major advantage over all other tests described if sensitivity and specificity to HIT are proven. Another recently described test employs a solid-phase adherence method to demonstrate the presence of heparin-dependent antibody.
Following the demonstration of antibodies against the H-PF4 complex in the serum of patients with HIT II, the ELISA technique for the detection of these antibodies was introduced.[46,49,50] Patient serum is incubated with the H-PF4 complex, and the presence of antibodies is detected with a secondary antibody conjugated with peroxidase or alkaline phosphatase. The ELISA showed a good correlation with the SRA procedure,[78,81,88] but comparison with the aggregation method was less reliable.[78,81,89] The ELISA method is characterized by greater sensitivity and reproducibility than the functional tests, and this procedure is technically easier to perform. However, the ELISA has demonstrated questionable specificity, in that anti-H-PF4 antibodies are detected in heparin-treated patients who did not present thrombocytopenia and in most patients undergoing heart surgery.[31,35,51,62,64,90-92] More importantly, this test was negative in some patients with HIT confirmed clinically or by positive functional tests.[78,81,88] It has been suggested that HIT II only occurs with high antibody titers and after persistent exposure to heparin,[64,90,93] and also that antigens different from the H-PF4 complex can be involved in the pathogenesis. In addition, assays from different manufacturers have different sensitivities and specificities.
In general, however, aside from the varying sensitivity levels of the methods and the lack of standardization, ELISA tests have proven to be predictive of the diagnosis of HIT II.[64,90] Therefore, especially in the absence of a highly suggestive clinical picture, it appears appropriate to support the clinical diagnosis with a functional test, as well as with measurement of the anti-H-PF4 antibody titer by ELISA.[48,79,82,87-89,96]
The best therapeutic strategy for patients with HIT II is not established, but reasonable guidelines have a wide consensus (Table 7). If HIT II is clinically probable, heparin therapy must be discontinued immediately, even in the absence of a confirmatory laboratory test. Platelet transfusion is contraindicated because it may worsen the thrombotic picture. Anticoagulant therapy with vitamin K antagonists should be initiated 3 or 5 days after heparin suspension, when platelet counts are increasing, but preferably before resolution of the thrombocytopenia to avoid potential worsening of the thrombotic picture.[6,97] However, heparin discontinuation alone and substitution with dicumaroids do not prevent the onset of severe thrombotic complications in nearly 50% of affected patients.[8,34,97] On the basis of these disappointing results, new approaches have been proposed that have included the use of LMWH, heparinoids, anticoagulating agents such as ancrod, prostaglandin (Iloprost), antiplatelet drugs, and thrombin inhibitors (argatroban and hirudin).
Table 7. Therapeutic Treatments for Patients With Heparin-Induced Thrombocytopenia (HIT)
Therapy contraindicated for patients with HIT Platelet transfusion Low-molecular-weight heparin Primary Discontinue heparin Substitute anticoagulant therapy Antithrombin drugs (argatroban, hirudin) Heparinoid (if negative laboratory test) Immunoglobulin Plasmapheresis Vitamin K antagonist (3-5 days after heparin cessation with increasing platelets) New approaches (preliminary) Antiplatelet drugs (GPIIb/IIIa inhibitors) Antithrombin agents (argatroban, hirudin) Pentasaccharide Secondary Ancrod Thrombolytics Vena cava filter Prostaglandin Thrombectomy Immune suppression
Low-molecular-weight heparins, heparinoids, ancrod, argatroban, and hirudin have been used in a significant number of patients.[4-6,9,75,98-100] A small clinical trial was conducted with ancrod, a viper-derived venom with anticoagulant action that showed efficacy. Other reports describe the use of plasmapheresis to remove immune complexes, or high doses of immunoglobulin alone or associated with LMWH and a heparinoid. Also indicated for use in patients with HIT II are thrombolytic agents; the insertion of filters in the inferior vein cava; and, in the case of arterial thrombosis, with limb ischemia, embolectomy, or thrombectomy.[4-6,92]
The rationale for the use of LMWHs in HIT II resided in the diminished interaction with PF4 of these heparins with decreased molecular weight and degree of sulfation.[51,98,105] However, the cross-reactivity with heparin-induced antibodies in vitro was shown to range from 60% to 100%.[4,51,81,97,106,107] Low-molecular-weight heparins should not be administered to patients with heparin antibody unless the absence of cross-reactivity has been demonstrated by an in vitro test. Nonetheless, some reports describe cases in which the use of LMWH was efficacious in controlling HIT even though cross-reactivity with heparin had been evidenced.[104,108]
In a study from our group, 2 synthetic pentasaccharides (SR90107A/Org31540 and SanOrg34006), which are in clinical development for the prophylaxis of postsurgical deep vein thrombosis, were tested in comparison to heparin and an LMWH (enoxaparin) for their relative platelet activation potential in HIT assays. Sera from patients with HIT II (n = 25), validated for heparin-dependent aggregation responses, and antibodies purified by H-PF4-sepharose column separation were used to study the effects of the 4 drugs using platelet aggregation. At comparable concentrations, heparin and enoxaparin consistently produced platelet activation (Table 8), whereas both pentasaccharides failed to produce a response at concentrations up to 100 U/mL (~50 [micro]mol/L). Similarly, in the SRA and flow cytometric assays, both heparin and enoxaparin produced positive responses, whereas the 2 pentasaccharides consistently failed to produce any effect.
Table 8. Heparin-Induced Thrombocytopenia (HIT) Sera-Mediated Platelet Activation Determined by Functional Assays of HIT(*)
% Platelet % Serotonin Agents Aggregation Release Heparin, U/mL Saline 12 [+ or -] 3 68 [+ or -] 8 0.1 54 [+ or -] 10 76 [+ or -] 6.2 100 15 [+ or -] 4 5 [+ or -] 3 Enoxaparin, U/mL 0.1 45 [+ or -] 6 80 [+ or -] 9 100 11 [+ or -] 3.9 49 [+ or -] 7 Pentasaccharide, U/mL([dagger]) 1.0 16 [+ or -] 3 7 [+ or -] 2 10 18 [+ or -] 2 8 [+ or -] 4 100 19 [+ or -] 4 7 [+ or -] 3 % P-Selectin % Microparticle Agents Expression Formation Heparin, U/mL Saline 5 [+ or -] 2 4.1 [+ or -] 3 0.1 50 [+ or -] 7 24 [+ or -] 17 100 10 [+ or -] 2.5 8 [+ or -] 3.5 Enoxaparin, U/mL 0.1 45 [+ or -] 8 30 [+ or -] 9 100 12 [+ or -] 4 9 [+ or -] 3 Pentasaccharide, U/mL([dagger]) 1.0 6 [+ or -] 2 4 [+ or -] 1 10 5 [+ or -] 2 10 [+ or -] 3 100 7 [+ or -] 3 5 [+ or -] 2
(*) Data represent mean percent [+ or -] SEM responses of 25 HIT patient sera. Similar results were observed with 2 different pentasaccharides (see reference 109).
We have further shown that in patients from a clinical trial substudy in which pentasaccharide was administered for the prophylaxis of deep vein thrombosis after hip surgery, no anti-H-PF4 antibody was detected during the treatment period (n = 10). However, in a comparable study with enoxaparin, 6 of 20 patients without clinical thrombocytopenia showed a positive anti-H-PF4 antibody titer. The observations from these studies suggest that the pentasaccharides with highly selective anti-Xa activity are devoid of generating anti-H-PF4 antibody, do not produce HIT responses in the presence of antibody, and may inhibit active HIT antibody platelet activation.
The major reported experiences of HIT treatment concern danaparoid sodium (Org10172, danaparoid, Lomoparan; Organon, Oss, The Netherlands), a mixture containing heparan sulfate (85%), dermatan sulfate (10%), and chondroitin sulfate (5%), whose cross-reactivity with heparin in vitro is less than 10%.[9,33,48] More than 600 patients with HIT II have been treated successfully with this drug, with a remarkable reduction in mortality due to thrombotic complications and in overall mortality in patients treated early.[9,75,104,110,111] However, cases of failure of treatment[106,112] or of danaparoid-induced fatal thrombotic thrombocytopenia have also been reported. In particular, treatment with danaparoid resolved thrombocytopenia in 91% of cases and significantly reduced mortality due to thrombotic complications of HIT from 28% to 5%, but did not reduce total mortality, which was 20%.[9,76] Danaparoid has been approved for the treatment of HIT II in New Zealand, Denmark, Luxembourg, Belgium, and Portugal.
Direct thrombin inhibitors are indicated for the treatment of HIT II. The first large-scale clinical trial of HIT patients treated with a thrombin inhibitor used argatroban, a thrombin inhibitor based on the structure of L-arginine. A number of patients with HIT II-associated thrombosis requiring angioplasty were also successfully treated with argatroban.[100,115] Hirudin, another thrombin inhibitor, was evaluated for use in the treatment of HIT II (mostly in Germany), and it was found to be efficacious.[116,117]
A further theoretical possibility, especially for patients with severe thrombotic complications refractory to thrombin inhibitor treatment alone, is the use of antiplatelet agents.[4-6] It has recently been demonstrated by several in vitro studies that antagonists of GPIIb/IIIa (Abciximab, Tirofiban, and Eptifibatide) are able to inhibit platelet aggregation induced by the serum of patients with HIT II.[86,118-121] The in vitro inhibition by GPIIb/IIIa inhibitors was more effective than inhibition of platelet activation by aspirin, as shown by SRA, platelet aggregation, and flow cytometry assays. The clinical usefulness of this treatment, combined with low-dose antithrombin agents, has shown preliminary beneficial results.[118,119]
Recently, we proposed that the prevalence of HIT antibodies in patients treated with immunosuppressive agents (such as cyclosporine) would be lower than in nontreated patients. Cyclosporine is used to suppress the immune systems of transplant recipients to prevent the production of antibodies against the foreign major histocompatibility factor of the donor organ, thus reducing the incidence of rejection. In testing the anti-H-PF4 antibody levels in cardiac surgery patients (n = 48) and cardiac transplant patients (n = 30), 23% of the cardiac surgery patients had positive antibody titers in contrast to 10% of transplant patients (Table 9). Of the positive cases, 6.3% of the cardiac surgery patients were positive by SRA, but none of the transplant patients were positive by SRA. Patients with rheumatoid arthritis (n = 9) and antiphospholipid syndrome (n = 21), which was treated with heparin and immunosuppressive therapy, did not exhibit a positive anti-H-PF4 antibody titer. These observations suggest that patients at high risk may be prophylactically treated with mild immunosuppression prior to heparinization to minimize the risk of HIT II. Clinical investigations are needed to validate this hypothesis.
Table 9. Heparin-Induced Thrombocytopenia Assay Results in Cardiac Surgery and Cardiac Transplant Patients Receiving Cyclosporine
Positive Antibody No. of Titer(*) Group Patients (% Prevalence) Cardiac surgery 48 11 (22.9) Cardiac transplant 30 3 (10.0) SRA([dagger])-Positive Group (% Prevalence) Cardiac surgery 3 (6.3) Cardiac transplant 0 (0)
(*) Diagnostica Stago HPIA-ELISA; P < .01 cardiac surgery vs cardiac transplant (ELISA data).
([dagger]) SRA indicates [sup.14]C-serotonin release assay.
The pathophysiology of HIT is now known to be mediated by antibodies to the anti-H-PF4 complex. These antibodies represent a heterogeneous group of IgG, IgA, and IgM antibodies that are generated in response to the neoepitope formed by the complex formation of heparin and PF4. Their functional form is capable of interacting with Fc receptors to activate platelets and endothelial cells. Cytokines are generated during this process as well. Activated platelets are consumed to form localized thrombi, resulting in the white clot syndrome. Inflammation plays an important role in the overall pathophysiology of HIT, and the elevated circulating levels of inflammatory markers have been found in patients with HIT.
The clinical diagnosis of this complex syndrome is based on platelet counts and, in extreme cases, the identification of characteristic purplish lesions on the skin. The laboratory diagnosis of HIT can be accomplished by using immunologic, platelet aggregation, and serotonin-release assays; however, the diagnostic efficacy of these tests is variable.
Patients with HIT can receive alternative anticoagulation therapy with several different drugs; however, only antithrombin drugs, such as argatroban and hirudin, are approved for this indication. Ancrod, a snake venom; danaparoid, a depolymerized mixture of glycosaminoglycans; and nonheparin glycosaminoglycans (dermatans) have also been used with success for alternative anticoagulation therapy. Plasmapheresis has been used for the management of HIT as well. Currently, synthetic pentasaccharides are being developed clinically, and the initial data are strongly suggestive of antithrombotic efficacy without any heparin antibody-related complications. Low-molecular-weight heparins are not indicated for the management of these patients. It has been shown that the activation of platelets during the acute HIT syndrome is not fully controllable by anticoagulant medications. Antiplatelet drugs, especially GPIIb/IIIa receptor antagonists, have been found to produce therapeutic effects and control the platelet-mediated pathophysiologic mechanisms more effectively than thrombin inhibitors.
Although we have made progress in better understanding the pathophysiology of HIT and have found better therapeutic options, there remain unsettled diagnostic and treatment issues that need to be addressed.
[1.] Davey MG, Lander H. Effect of injected heparin on platelet levels in man. J Clin Pathol. 1968;2:55-59.
[2.] Zucker MB. Biological aspects of heparin action: heparin and platelet function. Fed Proc. 1977;36:47-49.
[3.] Fabris F, Fussi F, Casonato A, et al. Normal and low molecular weight heparins: interaction with human platelets. Eur J Clin Invest. 1983;13:135-139.
[4.] Fondu P. Heparin-associated thrombocytopenia. Acta Clin Belg. 1995;50: 343-357.
[5.] Chong BH. Heparin-induced thrombocytopenia. Br J Haematol. 1995;89: 431-439.
[6.] Warkentin TE, Chong BH, Greinacher A. Heparin-induced thrombocytopenia: toward consensus. Thromb Haemost. 1998;79:1-7.
[7.] Chong BH. Heparin-induced thrombocytopenia. Aust N Z J Med. 1992;22: 145-152.
[8.] Warkentin TE, Kelton JG. A 14-year study of heparin-induced thrombocytopenia. Am J Med. 1996;101:502-507.
[9.] Magnani HN. Heparin-induced thrombocytopenia (HIT): an overview of 230 patients treated with Orgaran (Org10172). Thromb Haemost. 1993;70:554-561.
[10.] Schmitt BP, Adelman B. Heparin-associated thrombocytopenia: a critical review and pooled analysis. Am J Med Sci. 1993;305:208-215.
[11.] Nelson JC, Lerner RG, Goladstein R, Cagin NA. Heparin-induced thrombocytopenia. Arch Intern Med. 1978;138:548-552.
[12.] Powers PJ, Cuthbert D, Hirsh J. Thrombocytopenia found uncommonly during heparin therapy. JAMA. 1979;241:2396-2397.
[13.] Powers PJ, Kelton JG, Carter CJ. Studies of the frequency of heparin-associated thrombo-cytopenia. Thromb Res. 1984;33:439-443.
[14.] Eika C, Godal HC, Laake K, Hamborg T. Low incidence of thrombocytopenia during treatment with hog mucosa and beef lung heparin. Scand J Haematol. 1980;25:19-24.
[15.] Ansell J, Stepchuk N, Kumar R, Lopez A, Southard L, Deykin D. Heparin-induced thrombocytopenia: a prospective study. Thromb Haemost. 1980;43:61-65.
[16.] Kwaan HC, Kampmeior PA, Gomez HZ. Incidence of thrombocytopenia during therapy with bovine lung and porcine gut heparin preparations. Thromb Haemost. 1981;46:A680.
[17.] Weitberg AB, Spremulli E, Cummings FJ. Effect of low dose heparin on the platelet count. South Med J. 1982;75:190-192.
[18.] Bailey RT Jr, Ursick JA, Heim KL, Hilleman DE, Reich JW. Heparin-associated thrombocytopenia: a prospective comparison of bovine lung heparin, manufactured by a new process, and porcine intestinal heparin. Drug Intell Clin Pharm. 1986;20:374-378.
[19.] Bell WR, Tomasulo PA, Alving BM, Duffy TP. Thrombocytopenia occurring during the administration of heparin: a prospective study in 52 patients. Ann Intern Med. 1976;85:155-160.
[20.] Bell WR, Royall RM. Heparin-associated thrombocytopenia: a comparison of three heparin preparations. N Engl J Med. 1980;303:902-907.
[21.] Malcolm ID, Wigmore TA. Thrombocytopenia induced by low-dose subcutaneous heparin. Lancet. 1978;1:44.
[22.] Malcolm ID, Wigmore TA, Steinbrecher UP. Heparin-associated thrombocytopenia: low frequency in 104 patients treated with heparin of intestinal mucosal origin. Can Med Assoc J. 1979;120:1086-1088.
[23.] Holm HA, Eika C, Laake K. Thrombocytes and treatment with heparin from porcine mucosa. Scand J Haematol. 1980;36(suppl):81-84.
[24.] Gallus AS, Goodal KT, Bewick W, Chesterman CN. Heparin-associated thrombocytopenia: a case report and prospective study. Aust N Z J Med. 1980; 10:25-31.
[25.] Romeril KR, Anzimilt CMH, Hamer JW, Heaton DC. Heparin-induced thrombocytopenia: a case report and a prospective study. N Z Med J. 1982;95: 267-269.
[26.] Cipolle RJ, Rodvold KA, Seifert R, Clarens R, Ramirez-Lassepas M. Heparin-associated thrombocytopenia: a prospective evaluation of 211 patients. Ther Drug Monit. 1983;5:205-211.
[27.] Green D, Martin GJ, Shoichet SH. Thrombocytopenia in a prospective, randomised, double-blind trial of bovine and porcine therapy. Am J Med Sci. 1984;288:60-64.
[28.] Jonson RA, Lazarus KH, Henry DH. Heparin-induced thrombocytopenia: a prospective study. Am J Hematol. 1984;17:349-353.
[29.] Ansell JE, Price JM, Shan S, Beckner RR. Heparin-induced thrombocytopenia: what is its real frequency? Chest. 1985;66:878-882.
[30.] Kakkasseril JS, Cranley JJ, Panke T, Grannan K. Heparin-induced thrombocytopenia: a prospective study of 142 patients. J Vasc Surg. 1985;2:382-384.
[31.] Ayars GH, Tikoff G. Incidence of thrombocytopenia in medical patients on "mini-dose" heparin prophylaxis [letter to editor]. Am Heart J. 1980;99:816.
[32.] Warkentin TE. Heparin-induced thrombocytopenia: a clinicopathologic syndrome. Thromb Haemost. 1999;82:439-447.
[33.] Fabris F, Cordiano I, Luzzatto G, Cella G, Girolami A. Heparin-induced thrombocytopenia: prevalence in a large cohort of patients and confirmed role of PF4-heparin complex as the main antigen for antibodies. Clin Appl Thromb Hemost. 1997;3:203-209.
[34.] Wallis DE, Workman DL, Lewis BE, Steen L, Pifarre R, Moran JR Failure of early heparin cessation as treatment of heparin-induced thrombocytopenia. Am J Med. 1999;106:629-635.
[35.] Kappers-Klunne MC, Boon DMS, Hor WCJ, et al. Heparin-induced thrombocytopenia and thrombosis: a prospective analysis of the incidence in patients with heart and cerebrovascular diseases. Br J Haematol. 1997;96:442-446.
[36.] Levy G, Levy PY, Jamet M, Toroyan P. Thrombocytopenia due to a low molecular weight heparin during treatment with hypotensive and diuretics drugs. Ann Fr Anesth Reanim. 1991;10:586-588.
[37.] Elalamy I, Potevin F, Lecrubier C, Bara L, Marie JM, Samama MM. A fatal low molecular weight heparin-associated thrombocytopenia after hip surgery: possible usefulness of PF4-heparin ELISA test. Blood Coagul Fibrinolysis. 1997; 7:665-671.
[38.] Warkentin TE, Levine MN, Hirsh J, et al. Heparin-induced thrombocytopenia in patients treated with low molecular weight or unfractionated heparin. N Engl J Med. 1995;332:1330-1335.
[39.] Rhodes GR, Dixon RH, Silver D. Heparin-induced thrombocytopenia with thrombotic and hemorrhagic manifestation. Surg Gynecol Obstet. 1973;136:409-416.
[40.] Green D, Harris K, Reynolds N, Roberts M, Patterson R. Heparin immune thrombo-cytopenia: evidence for a heparin-platelet complex as the antigenic determinant. J Lab Clin Med. 1978;91:167-175.
[41.] Greinacher A, Michels I, Mueller-Eckhardt C. Heparin-associated thrombocytopenia: the antibody is not heparin specific. Thromb Haemost. 1992;67: 545-549.
[42.] Greinacher A, Michels I, Liebenhoff P, Presek P, Mueller-Echkardt C. Heparin-associated thrombocytopenia: immune complexes are attached to the platelet membrane by the negative charge of highly sulphated oligosaccharides. Br J Haematol. 1993;84:711-716.
[43.] Kelton JG, Smith JW, Warkentin TE, Hayward CPM, Denomma GA, Horsewood P. Immuno-globulin G from patients with heparin-induced thrombocytopenia binds to a complex of heparin and platelet factor 4. Blood. 1994;83:3232-3239.
[44.] Gruel Y, Boizard-Boval B, Wautier JL. Further evidence that alpha-granule components such as platelet factor 4 are involved in platelet-IgG-heparin interactions during heparin-associated thrombocytopenia. Thromb Haemost. 1993;70: 374-375.
[45.] Lynch DM, Howe SE. Heparin-associated thrombocytopenia: antibody binding specificity to platelet antigens. Blood. 1985;5:1176-1181.
[46.] Amiral J, Bridey F, Dreyfus M, et al. Platelet factor 4 complexed to heparin is the target for antibodies in heparin-induced thrombocytopenia. Thromb Haemost. 1992;68:95-96.
[47.] Amiral J, Marfaing-Koka M, Poncz M, Meyer D. The biological basis of immune heparin-induced thrombocytopenia. Platelets. 1998;9:77-91.
[48.] Greinacher A, Potzsch B, Amiral J, Dummel V, Eichner A, Mueller-Eckhardt C. Heparin-associated thrombocytopenia: isolation of the antibody and characterization of a multi-molecular PF4-heparin complex as the major antigen. Thromb Haemost. 1994;71:247-251.
[49.] Amiral J, Bridey F, Wolf M, et al. Antibodies to macromolecular platelet factor 4-heparin complexes in heparin-induced thrombocytopenia: a study of 44 cases. Thromb Haemost. 1995;73:21-28.
[50.] Visentin GP, Ford SE, Scott JP, Aster RH. Antibodies from patients with heparin-induced thrombocytopenia/thrombosis are specific for platelet factor 4 complexed with heparin or bound to endothelial cells. J Clin Invest. 1994;93:81-88.
[51.] Walenga JM, Koza MJ, Lewis BE, Pifarre R. Relative heparin-induced thrombocytopenic potential of low molecular weight heparins and new antithrombotic agents. Clin Appl Thromb Hemost. 1996;2(suppl 1):S21-S27.
[52.] Greinacher A. Antigen generation in heparin-associated thrombocytopenia: the non-immunologic type and the immunologic type are closely linked in their pathogenesis. Semin Thromb Hemost. 1995;21:106-116.
[53.] Baglin TP. Heparin-induced thrombocytopenia syndrome (HIT): diagnosis and treatment. Platelets. 1997;8:72-74.
[54.] Adelman B, Sobel M, Fujimura Y, Ruggeri ZM, Zimmerman TS. Heparin-associated thrombocytopenia: observations on the mechanism of platelet aggregation. J Lab Clin Med. 1989;113:104-110.
[55.] Anderson CL, Chako GW, Osborne JM, Brandt JT. The Fc receptor for immunoglobulin G (Fc[Gamma]/RIIa) on human platelets. Semin Thromb Hemost. 1995; 21:1-9.
[56.] Kelton JG, Sheridan D, Sanots A, et al. Heparin-induced thrombocytopenia: laboratory studies. Blood. 1988;723:925-930.
[57.] Chong BH, Fawaz I, Chesterman CN, Berndt MC. Heparin-induced thrombocytopenia: mechanism of interaction of the heparin-dependent antibody with platelets. Br J Haematol. 1989;73:235-240.
[58.] Burgess JK, Lindeman R, Chesterman CN, Chong BH. Single amino acid mutation of Fc[Gamma] receptor is associated with the development of heparin induced thrombocytopenia. Br J Haematol. 1995;91:761-766.
[59.] Arepally G, McKenzie SE, Jiang XM, Poncz M, Cines DB. Fc[Gamma]RIIa His/Arg 131 polymorphism subclass-specific IgG anti-heparin/PF4 antibodies and clinical course in patients with heparin-induced thrombocytopenia and thrombosis. Blood. 1997;89:370-375.
[60.] Horne KM, Hutchinson KJ. Simultaneous binding of heparin and platelet factor-4 to platelets: further insights into mechanism of heparin-induced thrombocytopenia. Am J Hematol. 1998;58:24-30.
[61.] Suh JS, Malik MI, Aster RH, Visentin GP. Characterization of the humoral immune response in heparin-induced thrombocytopenia. Am J Hematol. 1997; 54:196-201.
[62.] Amiral J, Peynaud-Debayle E, Wolf M, Bridey F, Vissac AM, Meyer D. Generation of antibodies to heparin-PF4 complexes without thrombocytopenia in patients treated with unfractionated or low-molecular weight heparin. Am J Hematol. 1996;52:90-95.
[63.] Amiral J, Wolf M, Fischer AM, Boyer-Neumann C, Vissac AM, Meyer D. Pathogenicity of IgA and/or IgM antibodies to heparin-PF4 complexes in patients with heparin-induced thrombocytopenia. Br J Haematol. 1996;92:954-959.
[64.] Visentin GP. Heparin-induced thrombocytopenia: molecular pathogenesis. Thromb Haemost. 1999;82:448-456.
[65.] Blank M, Cines DB, Arepally G, Eldor A, Afek A, Shoenfeld Y. Pathogenicity of human anti-platelet factor 4 (PF4)/heparin in vivo: generation of mouse anti-PF4/heparin and induction of thrombocytopenia by heparin. Clin Exp Immunol. 1997;108:333-339.
[66.] Ahmad S, Walenga JM, Jeske WP, et al. Lack of thrombocytopenic response after low molecular weight heparins usage is due to the generation of non-functional anti-heparin-platelet factor 4 antibodies [abstract]. Blood. 1999;94(suppl 1):624a.
[67.] Ahmad S, Walenga JM, Jeske WP, Cella G, Fareed J. Functional heterogeneity of anti-heparin-platelet factor 4 antibodies: implications in the pathogenesis of the HIT syndrome. Clin Appl Thromb Hemost. 1999;5(suppl 1):S32-S37.
[68.] Fareed J, Walenga JM, Hoppensteadt DA, et al. Soluble adhesion molecules in the HIT syndrome: pathophysiologic role and therapeutic modulation. Clin Appl Thromb Hemost. 1999;5(suppl 1):S38-S44.
[69.] Vermylen J, Hoylaerts MF, Arnout J. Antibody-mediated thrombosis. Thromb Haemost. 1997;78:420-426.
[70.] Mikhailidis DP, Jagroop IA, Ganotakis ES. Pathophysiology of heparin-induced thrombocytopenia (HIT). Platelets. 1997;8:66-67.
[71.] Boshkov L, Warkentin TE, Hayward CPM, Andrew M, Kelton JG. Heparin-induced thrombocytopenia and thrombosis: clinical and laboratory studies. Br J Haematol. 1993;84:322-328.
[72.] Sallah S, Thomas P, Roberts HR. Warfarin and heparin-induced skin necrosis and the purple toe syndrome: infrequent complications of anticoagulant treatment. Thromb Haemost. 1997;78:785-790.
[73.] Nand S, Wong W, Yuen B, Yetter A, Schmulbach E, Fisher SG. Heparin-induced thrombocytopenia with thrombosis: incidence, analysis of risk factors, and clinical outcomes in 108 consecutive patients treated in a single institution. Am J Hematol. 1997;56:12-16.
[74.] Hach-Wunderle V, Kainer K, Krug B, Mueller-Berghaus G, Potzsch B. Heparin-associated thrombosis despite normal platelet counts. Lancet. 1994;344: 469-470.
[75.] Weismann RE, Tobin RW. Arterial embolism occurring during systemic heparin therapy. Arch Surg. 1958;76:219-224.
[76.] Magnani HN. Orgaran (danaparoid sodium) use in the syndrome of heparin-induced thrombocytopenia. Platelets. 1997;8:74-81.
[77.] Sheridan D, Carter C, Kelton JG. A diagnostic test for heparin-induced thrombocytopenia. Blood. 1986;67:27-30.
[78.] Greinacher A, Amiral J, Dummel V, Vissac A, Kiefel V, Mueller-Eckhardt C. Laboratory diagnosis of heparin-associated thrombocytopenia and comparison of platelet aggregation test, heparin-induced platelet activation test, and platelet factor 4/heparin enzyme-linked immunosorbent assay. Transfusion. 1994;34:381-385.
[79.] Warkentin TE, Hayward CPM, Smith CA, Kelly PM, Kelton JG. Determinants of donor platelet variability when testing for heparin-induced thrombocytopenia. J Lab Clin Med. 1992;120:371-379.
[80.] Walenga JM, Jeske WP, Fasanella AR, Wood JJ, Ahmad S, Bakhos M. Laboratory diagnosis of heparin-induced thrombocytopenia. Clin Appl Thromb Hemost. 1999;5(suppl 1):S21-S27.
[81.] Walenga JM, Jeske WP, Wood JJ, Ahmad S, Lewis BE, Bakhos M. Laboratory tests for heparin-induced thrombocytopenia: a multicenter study. Semin Hematol. 1999;36(suppl 1):22-28.
[82.] Greinacher A, Michels I, Kiefel V, Mueller-Eckhardt C. A rapid and sensitive test for diagnosing heparin-induced thrombocytopenia. Thromb Haemost. 1991; 66:734-736.
[83.] Amiral J. Diagnostic tests in heparin-induced thrombocytopenia. Platelets. 1997;8:68-72.
[84.] Teitel JM, Gorss P, Blake P, Garvey MB. A bioluminescent adenosine nucleotide release assay for the diagnosis of heparin-induced thrombocytopenia. Thromb Haemost. 1996;76:479-480.
[85.] Tomer A. A sensitive and specific functional flow cytometric assay for the diagnosis of heparin-induced thrombocytopenia. Br J Haematol. 1997;98:648-656.
[86.] Jeske WP, Walenga JM, Szatkowski E, et al. Effect of glycoprotein IIb/IIIa antagonists on the HIT serum-induced activation of platelets. Thromb Res. 1997; 88:271-281.
[87.] Leach MF, Cooper LK, AuBuchon JP. Detection of drug-dependent, platelet-reactive antibodies by solid-phase red cell adherence assays. Br J Haematol. 1997;97:755-761.
[88.] Arepally G, Reynolds C, Tomanski A, et al. Comparison of PF4/heparin ELISA assay with the [sup.14]C-serotonin release assay in the diagnosis of heparin-induced thrombocytopenia. Am J Clin Pathol. 1995;104:648-654.
[89.] Look KA, Sahud M, Flaherty S, Zehnder J. Heparin-induced platelet aggregation vs. platelet factor 4 enzyme-linked immunosorbent assay in the diagnosis of heparin-induced thrombocytopenia-thrombosis. Am J Clin Pathol. 1997; 108:78-82.
[90.] Bauer TL, Arepally G, Konkle BA, et al. Prevalence of heparin-associated antibodies without thrombosis in patients undergoing cardiopulmonary bypass surgery. Circulation. 1997;95:1242-1246.
[91.] Walenga JM, Jeske WP, Fasanella AR, Wood JJ, Bakhos M. Laboratory tests for the diagnosis of heparin-induced thrombocytopenia. Semin Thromb Hemost. 1999;25(suppl 1):43-49.
[92.] Walenga JM, Lewis BE, Hoppensteadt DA, Fareed J, Bakhos M. Management of heparin-induced thrombocytopenia and heparin-induced thrombocytopenia and thrombosis syndrome. Clin Appl Thromb Hemost. 1997;3(suppl 1): S53-S63.
[93.] Kelton JG, Warkentin TE. Heparin-induced thrombocytopenia: what the serologists have taught us. J Lab Clin Med. 1996;128:346-368.
[94.] Amiral J, Marfaing-Koka A, Wolf M, et al. Presence of autoantibodies to interleukin 8 or neutrophil-activating peptide-2 in patients with heparin-associated thrombocytopenia. Blood. 1996;88:410-416.
[95.] Izban KF, Lietz HW, Hoppensteadt DA, et al. Comparison of two PF4/ heparin ELISA assays for the laboratory diagnosis of heparin-induced thrombocytopenia. Semin Thromb Hemost. 1999;25(suppl 1):51-56.
[96.] Nguyen P, Droulle C, Potron G. Comparison between platelet factor 4/ heparin complexes ELISA and platelet aggregation test in heparin-induced thrombocytopenia. Thromb Haemost. 1995;74:804-805.
[97.] Wallis DE, Quintos R, Wehrmacher W, Messmore H. Safety of warfarin anticoagulation on patients with heparin-induced thrombocytopenia. Chest. 1999;116:1333-1338.
[98.] Keeling DM, Richards EM, Baglin TP. Platelet aggregation in response to four low molecular weight heparins and the heparinoid ORG10172 in patients with heparin-induced thrombocytopenia. Br J Haematol. 1994;86:425-426.
[99.] Schiele F, Vuillemenot A, Kramarz P, et al. Use of recombinant hirudin as antithrombotic treatment in patients with heparin-induced thrombocytopenia. Am J Hematol. 1995;50:20-25.
[100.] Lewis BE, Walenga JM, Wallis DE. Anticoagulation with Novastan (argatroban) in patients with heparin-induced thrombocytopenia and heparin-induced thrombocytopenia and thrombosis syndrome. Semin Thromb Hemost. 1997;23:197-202.
[101.] Demers C, Ginsberg JS, Brill-Edwards P, et al. Rapid anticoagulation using ancrod for heparin-induced thrombocytopenia. Blood. 1991;78:2194-2197.
[102.] Bouvier JL, Lefebre P, Villain P, et al. Treatment of serious heparin induced thrombocytopenia by plasma exchange: report of 4 cases. Thromb Res. 1988;51: 355-356.
[103.] Frame JN, Mulvey KP, Phares JC, Anderson MJ. Correction of severe heparin-induced thrombocytopenia with intravenous immunoglobulin. Ann Intern Med. 1989;111:946-947.
[104.] Harenberg J, Huhle G, Piazolo L, Wang LU, Heene DL. Anticoagulation in patients with heparin-induced thrombocytopenia type II. Semin Thromb Hemost. 1997;23:189-196.
[105.] Denton J, Lane DA, Thunberg L, Slater AM, Lindahl U. Binding of platelet factor 4 to heparin oligosaccharides. Biochem J. 1983;209:455-460.
[106.] Kikta M, Keller MP, Humphrey PW, Silver D. Can low molecular weight heparins and heparinoids be safely given to patients with heparin-induced thrombocytopenia syndrome? Surgery. 1993;114:705-710.
[107.] Haas S, Walenga JM, Jeske WP, Fareed J. Heparin-induced thrombocytopenia: clinical considerations of alternative anticoagulation with various glycosaminoglycans and thrombin inhibitors. Clin Appl Thromb Hemost. 1999;5: 52-59.
[108.] Luzzatto G, Cordiano I, Patrassi G, Fabris F. Heparin-induced thrombocytopenia: discrepancy between the presence of IgG cross-reacting in vitro with fraxiparine and its successful clinical use. Thromb Haemost. 1995;74:1607-1608.
[109.] Ahmad S, Jeske WP, Walenga JM, et al. Synthetic pentasaccharides do not cause platelet activation by anti-heparin-platelet factor 4 antibodies. Clin Appl Thromb Hemost. 1999;5:259-266.
[110.] Chong BH. Comment on "Failure of Orgaran therapy in a patient with a previous heparin-induced thrombocytopenia syndrome." Br J Haematol. 1995; 90:970.
[111.] Nurmohamed MT, ten Cate H, ten Cate JW. Low molecular weight heparin(oid)s: clinical investigation and practical recommendations. Drugs. 1997;53: 736-751.
[112.] Tardy-Poncet B, Mahul P, Beraud AM, Favre JP, Tardy B, Guyotat D. Failure of Orgaran therapy in a patient with a previous heparin-induced thrombocytopenia syndrome. Br J Haematol. 1995;90:969-970.
[113.] Tardy B, Tardy-Poncet B, Viallon A, Piot M, Mazet E. Fatal danaparoid-sodium induced thrombocytopenia and arterial thromboses. Thromb Haemost. 1998;80:530.
[114.] Lewis BE. Preliminary results of a prospective randomized controlled trial of argatroban vs. conventional therapy for heparin-induced thrombocytopenia. Presented at: Argatroban: A Novel Antithrombotic Drug for the Treatment of Heparin-Induced Thrombocytopenia Symposium; June 12, 1997; Florence, Italy.
[115.] Matthai WH. Use of argatroban during percutaneous coronary interventions in patients with heparin-induced thrombocytopenia. Semin Thromb Hemost. 1999;25(suppl 1):57-60.
[116.] Greinacher A, Volpel H, Janssens U, et al. Recombinant hirudin (Lepirudin) provides safe and effective anticoagulation in patients with heparin-induced thrombocytopenia: a prospective study. Circulation. 1999;99:73-80.
[117.] Beijering RJR, ten Cate H, ten Cate JW. Clinical applications of new antithrombotic agents. Ann Hematol. 1996;72:177-183.
[118.] Walenga JM, Jeske WP, Wallis DE, et al. Clinical experience with combined treatment of thrombin inhibitors and GPIIb/IIIa inhibitors in patients with HIT. Semin Thromb Hemost. 1999;25(suppl 1):77-81.
[119.] Jeske WP, Szatkowski E, Wood JJ, Messmore HL, Herbert J-M, Walenga JM. Inhibition of platelet activation in HIT: thrombin inhibitors vs. antiplatelet agents [abstract]. Blood. 1998;92(10):180a.
[120.] Greinacher A. Treatment of heparin-induced thrombocytopenia. Thromb Haemost. 1999;82:457-467.
[121.] Liem TK, Teel R, Shukla S, Silver D. The glycoprotein IIb/IIIa antagonist c7E3 inhibits platelet aggregation in the presence of heparin-associated antibodies. J Vasc Surg. 1997;25:124-130.
[122.] Fareed J, Hoppensteadt DA, Jeske P, Walenga JM, Ahmad S, Torri R. Immunosuppression results in a reduction in anti-heparin-platelet factor 4 antibodies: implications in the management of heparin-induced thrombocytopenia [abstract]. Blood. 1999;94(suppl 1):19a.
Accepted for publication July 13, 2000.
From the Department of Medical and Surgical Sciences, University of Padua, Medical School, Padua, Italy (Drs Fabris and Cella), and the Department of Pathology and Cardiovascular Institute, Loyola University Chicago, Stritch School of Medicine, Maywood, Ill (Drs Ahmad, Jeske, Walenga, and Fareed).
Reprints: Jawed Fareed, PhD, Loyola University Chicago, Stritch School of Medicine, 2160 S First Ave, 102/2652, Maywood, IL 60153.
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|Author:||Fabris, Fabrizio; Ahmad, Sarfraz; Cella, Giuseppe; Jeske, Walter P.; Walenga, Jeanine M.; Fareed, Ja|
|Publication:||Archives of Pathology & Laboratory Medicine|
|Date:||Nov 1, 2000|
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