Heparin-induced thrombocytopenia: Understanding improves but questions remain

Heparin-induced thrombocytopenia: Understanding improves but questions remain

Heparin-induced thrombocytopenia: Understanding improves but questions remain Abbreviations: Fc-yRII = crystallizable fragment-~ [of antigen); HITP = ...

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Heparin-induced thrombocytopenia: Understanding improves but questions remain Abbreviations: Fc-yRII = crystallizable fragment-~ [of antigen); HITP = heparin-induced thrombocytopenia; IgG = immunoglobulin G; PF4 = platelet factor 4

he occurrence of HITP, a common and sometimes devastating complication of heparin therapy, has been recognized as a distinct clinical entity for more than 30 years. 1 Observations made since 1980 have shown that serum from patients with HITP contains IgG antibodies that are capable of activating platelets in the presence of heparin at low concentrations (0.1 to 1 U/ml) but not at high concentrations (10 to 100 U/ml). 2'3 This reaction can be blocked by monoclonal antibodies reactive with FcyRII, the only Fc receptor known to be expressed on platelets. 4 On the basis of these findings it was thought for a number of years that antibodies associated with HITP were specific for heparin and reacted with the anticoagulant to form immune complexes that caused platelet activation and thrombocytopenia. The associated thrombosis was felt to be triggered by the procoagulant activity inherent in activated platelets and in micropartMes created by platelet fragmentation. 3'5 In general, attempts to develop a binding assay for measurement of HITP antibodies were unsuccessful. In the absence of such an assay, two tests dependent on platelet activation, the platelet aggregation test6 and the serotonin release test2 were relied on for diagnosis. Work done by several groups in the last few years has provided new clues to the pathogenesis of HITP. The salient observation is that the associated antibodies are specific not for heparin but for complexes of heparin and PF4, a heparin-binding platelet otgranule protein. 7-1° Visentin et al.8 directly demonstrated binding of these antibodies to resting platelets in the presence of optimal concentrations of heparin and PF4 and to heparin-like endothelial cell glycosaminoglycans in the presence of heparin alone and postulated that thrombocytopenia and thrombosis result from the following sequence of events: (1) injected heparin scavenges PF4 normally associated with endothelial cell glycosaminoglycans and releases additional PF4 by a direct action on circulating platelets, leading

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J Lab Clin Med 1996;127:418-9. Copyright © 1996by Mosby-YearBook, Inc. 0022-2143/96 $5.00 + 0 5/1/71887 418

to the formation of heparin:PF4 complexes; (2) certain of these complexes, formed at optimal heparin: PF4 ratios, bind antibody to form immune complexes; (3) immune complexes formed close to the platelet surface bind to platelet Fc receptors and cause platelet activation; (4) this leads to further PF4 release, formation of additional immune complexes, a cycle of platelet activation, and, ultimately, thrombocytopenia; (5) PF4 released in excess of the amount that can be neutralized by available heparin reassociates with glycosaminoglycan molecules on endothelium; (6) this creates targets on the vessel wall for antibody- and cell-mediated endothelial cell injury, placing the patient at risk for thrombosis. 8 The finding that heparin: PF4 complexes can be immobilized and used to detect HITP antibodies with high sensitivity opens the possibility that HITP can now be diagnosed quickly and specifically.7'8 Recent preliminary reports indicate that more than half of patients treated with heparin during cardiac bypass surgery produce antibodies detectable in this assay. 1~ Studies are now in progress to determine whether the heparin:PF4 enzyme-linked immunosorbent assay test will enable early identification of patients at risk for this condition. Horne and Alkins, 12 in this issue of the JOURNAL, provide information about the mechanism(s) by which HITP antibodies bind to platelets in the presence of heparin and PF4. They describe the binding of radiolabeled, partially purified HITP antibodies to platelets, which was maximal at molar heparin: PF4 ratios approximating 1:1. This reaction was unequivocal with thrombin-activated platelets but was weak and inconsistent with resting platelets. Surprisingly, it was found that F(ab')2 antibody fragments reacted with activated platelets about as well as intact IgG and that this binding was not blocked by the monoclonal antibody IV.3, which is specific for the platelet Fc receptor. These findings suggest that heparin:PF4 complexes form on the surface of platelets to provide a site for subsequent antibody binding, as suggested also by two other groups. 9'13 However, they are at variance with studies from our laboratory showing that monoclonal IV.3 ablates heparin: PF4-dependent binding of IgG to platelets,

J Lab Clin Med Volume ]27, Number 5

Editorial

which suggests that i m m u n e complexes containing heparin, PF4, and I g G are f o r m e d first in the fluid phase and then bind to platelet Fc receptors. T h e discrepancy may be explained in part by the use of thrombin-activated platelets by H o m e and Alkins and the use of resting platelets by our own group. This issue is of m o r e than academic importance, because platelet Fc receptors are capable of binding only IgG. Thus only I g G antibodies should be able to mediate binding of soluble i m m u n e complexes to platelets to cause activation and thrombocytopenia. If assembly of the complex occurs on the platelet surface, as suggested by H o m e and Alkins, IgM and I g A antibodies should also be able to bind and possibly p r o m o t e platelet injury. It should be kept in mind that none of the in vitro studies reported to date were done under conditions that mimic the in vivo situation in which resting, or partially activated, platelets circulate in a plasma milieu where they are subjected to shear stress and other factors that influence their state of activation. Further studies of the binding of H I T P antibodies to platelets under more "physiologic" conditions are therefore needed. N o r is it clear why some patients with antibodies specific for heparin:PF4 experience only mild thrombocytopenia when challenged with heparin while others have serious thromboembolic complications. Recent reports suggest that certain genetic polymorphisms may predispose patients to the more severe side effects of heparin sensitivity14'15; further studies of this possibility are urgently needed. Finally, we need to find ways to prevent this condition and eliminate its associated morbidity and mortality. A recent report indicating that H I T P is less likely to occur in patients treated with low-molecular-weight heparin than in those receiving unfractionated heparin is encouraging in this regard. 16 RICHARD H. ASTER, MD

Clinical Professor of Medicine and Pathology Medical College of Wisconsin President The Blood Center of Southeastern Wisconsin, Inc. REFERENCES

1. Weismann RE, Tobin RW. Arterial embolism occurring during systemic heparin therapy. Arch Surg 1958;76:219-27.

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2. Sheridan D, Carter C, Kelton JG. A diagnostic test for heparin-induced thrombocytopenia. Blood 1986;67:27-30. 3. Chong BH. Heparin-induced thrombocytopenia. Br J Haematol 1995;89:431-9. 4. Kelton JG, Sheridan D, Santos A, et al. Heparin-induced thrombocytopenia: laboratory studies. Blood 1988;72:92530. 5. Warkentin TE, Hayward CPM, Boshkov LK, et al. Sera from patients with heparin-induced thrombocytopenia generate platelet-derived microparticles with procoagulant activity: an explanation for the thrombotic complications of heparininduced thrombocytopenia. Blood 1994;84:3691-9. 6. Chong BH, Burgess J, Ismail F. The clinical usefulness of the platelet aggregation test for the diagnosis of heparin-induced thrombocytopenia. Thromb Haemost 1993;69:344-50. 7. Amiral J, Bridey F, Dreyfus M, et al. Platelet factor 4 complexed to heparin is the target for antibodies generated in heparin-induced thrombocytopenia [Letter]. Thromb Haemost 1992;68:95-6. 8. 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;93:81-8. 9. Kelton JG, Smith JW, Warkentin TE, Hayward CPM, Denomme GA, Horsewood P. Immunoglobulin G from patients with heparin-induced thrombocytopenia binds to a complex of heparin and platelet factor 4. Blood 1994;83:3232-9. 10. 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;71:247-51. 11. Visentin GP, Malik MI, Menden K, Aster RH. A prospective study of the formation of antibodies reactive with heparin: PF4 complexes in patients treated with heparin. Blood 1994; 84(suppl 1):80a. 12. Home MK III, Alkins BR. Platelet binding of IgG from patients with heparin-induced thrombocytopenia. J LABCLIN MED 1996;127:435-42. 13. Greinacher A. Antigen generation in heparin-associated thrombocytopenia: the nonimmunologic type and the immunologic type are closely linked in their pathogenesis. Semin Thromb Haemost 1995;21:106-16. 14. Burgess JK, Lindeman R, Chesterman CN, Chong BH. Single amino acid mutation of Fc-y receptor is associated with the development of heparin-induced thrombocytopenia. Br J Haematol 1995;91:761-6. 15. Gardyn J, Sorkin P, Kluger Y, et al. Heparin-induced thrombocytopenia and fatal thrombosis in a patient with activated protein C resistance. Am J Hematol 1995;50:292-5. 16. Warkentin TE, Levin MN, Hirsh J, et al. Heparin-induced thrombocytopenia in patients treated with low-molecularweight heparin or unfl'actionated heparin. N Engl J Med 1995:332;1330-5.