Glanzmann thrombasthenia

The ability of platelets to aggregate and to form a platelet plug is central to the maintenance of normal hemostasis. When platelets have normal function, the severity of bleeding is related to the degree of thrombocytopenia. In patients with normal platelet production, the most common cause of thrombocytopenia is due to immune mechanisms that results in platelet injury and removal from the circulation. These mechanisms involve the binding of platelet-associated immunoglobulins and are classified as immune. Immune thrombocytopenias can be caused by autoantibodies (autoimmune thrombocytopenia), alloantibodies (isoimmune thrombocytopenia), or drug-induced immune complexes and conditions secondary to autoimmune disorders such as systemic lupus erythematosus. In this paper the focus is on alloimmune thrombocytopenias resulting from the formation of alloantibodies to platelet specific antigens.

The clinical importance of the platelet alloantigens is due to their ability to elicit alloantibody production. Alloantigens, also referred to as isoantigens, are substances that induce the production of alloantibodies when they are infused into individuals of the same species who lack the specific alloantigen.

ABBREVIATIONS: ATP = alloimmune (isoimmune) thrombocytopenia purpura; GP = glycoprotein; GT = Glanzmann's thrombasthenia; HPA = human platelet alloantigen; ITP = immune thrombocytopenic purpura; NAIT = neonatal alloimmune thrombocytopenia; PAIg = platelet-associated immunoglobulins; PTP = post-transfusion (isoimmune) purpura; SLE = systemic lupus erythematosus; vWf = von Willebrand's factor.

INDEX TERMS: alloantibody; alloantigen; human platelet antigens; immune thrombocytopenic purpura; neonatal alloimmune thrombocytopenia; platelet glycoproteins.

The ability of platelets to aggregate and to form a platelet plug is central to the maintenance of normal hemostasis. For effective platelet plug formation platelets must be present in adequate numbers and have normal function. The normal reference range for platelet counts, which is usually stated as being 140 to 440 x 10^sup 9^/L, does not change significantly after 20 weeks of gestation through adulthood.1,2 Hemostatic disorders associated with platelet counts less than the reference range are classified as thrombocytopenias.3

When platelets have normal function, the severity of bleeding is related to the degree of thrombocytopenia. For platelet counts 50 x 10^sup 9^/L will have no hemorrhagic symptoms unless subjected to trauma or surgery. Platelet counts

Thrombocytopenia is the most common cause of abnormal bleeding.5 The development of thrombocytopenia can result from inadequate production, abnormal pooling, or increased destruction. In patients with normal platelet production, the most common cause of thrombocytopenia is due to an immune mechanism that results in platelet injury and removal from the circulation. These mechanisms involve the binding of platelet-associated immunoglobulins (PAIg) and are classified as immune.

IMMUNE THROMBOCYTOPENIAS

Immune thrombocytopenias can be caused by autoantibodies (autoimmune thrombocytopenia), alloantibodies (isoimmune thrombocytopenia), or drug-induced immune complexes and conditions secondary to autoimmune disorders such as systemic lupus erythematosus (SLE). Autoimmune disorders include acute and chronic immune thrombocytopenia purpura, SLE, and some drug associated immune thrombocytopenias. Neonatal alloimmune thrombocytopenia (NAIT), post-transfusion refractoriness, and post-transfusion purpura are examples of alloimmune thrombocytopenia.

In this paper the focus is on alloimmune thrombocytopenias resulting from the formation of alloantibodies to platelet specific antigens. Alloimmune (isoimmune) thrombocytopenia purpura (ATP) is the only common severe form of thrombocytopenia not caused by some other condition such as an infection.6

PLATELET ALLOANTIGENS

The clinical importance of platelet alloantigens is due to their ability to elicit alloantibody production. Alloantigens, also referred to as isoantigens, are substances that induce the production of alloantibodies when they are infused into individuals of the same species who lack the specific alloantigen. The ABO and Rh blood groups are examples of alloantigens on red blood cells.7 Infusion of type A blood into a patient who lacks the type A alloantigen can induce the production of antibodies and the destruction of the red blood cells. In the same way, infusion of platelets with alloantigens into a patient who lacks the antigen can induce an immunologic response.8 Platelet specific alloantigens that are associated with the platelet membrane are glycoproteins GPIa, GPIb, GPIIa, GPIIb, and GPIIIa. GPIV and GPV may also be antigenic.7

Glanzmanns Thrombasthenia (GT) is an example of a genetic condition where the patient's platelets lack HPA-la. GT patients have a qualitative or quantitative defect of the glycoprotein (GP) IIb-IIIa complex (allbb3 integrin).9 This IIb-IIIa complex is frequently called the fibrinogen receptor. This recessive autosomal bleeding disorder is characterized by abnormal platelet aggregation, due to a failure to bind fibrinogen when activated, and an abnormal clot retraction.'o Since 98% of Caucasians, and close to 100% of Asian and African American populations have HPA-la, GT patients who lack the HPA-la antigen are at great risk of developing alloantibodies when they are exposed to the HP-la antigen through transfusion or during pregnancy

Specific GPs act as receptors for factors important in hemostasis. As an example, GPIb-IX is important for binding von Willebrand's factor (vWf) and GPIIb-GPIIIa (a11b3 integrin) binds fibrinogen. Antibodies to these GP can inhibit the binding of vWf and fibrinogen.ll The severity of bleeding in immune disorders is associated with the degree of thrombocytopenia as well as inhibition of platelet function.

PLATELET ALLOANTIGEN NOMENCLATURE

One of the difficulties encountered when studying or reading about the platelet specific antigens is the variety of names given to the same antigen or epitope. Table 1 lists the platelet specific antigens using the three most commonly used nomenclature systems. This table is a modification of several tables.2,712-'7 It is hoped that this table will be helpful in sorting out some of the confusion that has resulted from the use of multiple names.

The platelet alloantigen name confusion is analogous to the problems of trying to understand coagulation when the clotting factors were refered to by several different names. In coagulation, the International Committee on Nomenclature of Blood Clotting made the decision to adopt the use of roman numerals to designate the different coagulation factors. This decision has been a great help in standardizing communication in the field. A criticism of the roman numeral designation is that it provides no information about the function of the coagulation factor.

Before 1990, naming of platelet alloantigens was based on the abbreviated surnames of the individuals in whom the antibody was first discovered. In some cases there were several surnames for the same antigen. As an example Brb, Hc, and Zave are three different names for the same platelet alloantigen. To standardize nomenclature, von dem Borne and Decary proposed a system where antigens were identified by a human platelet alloantigen (HPA) number.'5 The HPA numbers and the corresponding serologic designation of the antigen along with the location of the antigen are listed in Table 1. The two serological forms of the allele were designated as either a or b. As an example, the old serological designation of PI^'/ZWa is now referred to as HPA-la, and lI/ZWb is now HPA-lb. This system of nomenclature which is based on serological typing, has been introduced by the Platelet Serology Working Party.'5

One advantage of the HPA nomenclature is that it does not require a complete genotyping of an individual.l3 The major disadvantage is that it fails to take into account the molecular basis of the alloantigen's specificity. Molecular studies indicate that HPAla, HPA-4a, HPA-6a, HPA-7a, and HPA-8a are probably five different names for the same alloantigen." However, it should be noted that antibodies bind to a particular part of an antigen called the epitope or antigenic determinant. A particular antigen can have several epitopes. HPA-la, HPA-4a, HPA-6a, HPA-7a, and HPA8a designations probably identify five different epitopes on the same GPIIIa alloantigen. At this time there is no consensus on a standardized system for identifying human platelet alloantigens.18

Column one of Table 1 lists the HPA-number as proposed by von dem Borne.14 The old abbreviated surnames for the serologic designation are listed in the second column. The alloantigens on which the epitopes are localized are listed in column three. The molecular names or allelic forms of the different epitopes are listed in column four. The molecular name for HPA-1 can be written as GPIIIa-Leu33Pro.13 This means that HPA-la differs from HPAlb by only one amino acid at position 33. For HPA-la, the amino acid at position 33 is a leucine where as for HPA-lb it is a proline.

The phenotype and gene frequencies vary between ethnic groups. As an example, the phenotype frequency for HPA-1 in Caucasians is about 97.8%. In African Americans and in Asians the frequency is close to 100%. This information suggests that 2% of the Caucasian population could be at risk of developing alloantibodies to this antigen.12

NEONATAL IMMUNE THROMBOCYTOPENIA PURPURA

Platelet antigen incompatibility between the mother and fetus is the most common cause of neonatal alloimmune thrombocytopenia (NAIT). NAIT is analogous to Rh disease in the newborn in that thrombocytopenia in the fetus is due to maternal antibodies binding to the fetus's platelets. Transfer of fetal red blood cells and platelets to the maternal circulation can stimulate an immune response with production of immunoglobulin that can cross the placenta.19 The maternal IgG antibody production is elicited by the paternal antigen on the fetal platelets.zo Unlike hemolytic disease of the newborn due to the Rh factor, NAIT is evident in the first pregnancy in approximately 50% of the cases. This suggests that sensitization to the platelet antigen by previous pregnancies or transfusions is not required."

In pregnancy both auto- and alloimmune thrombocytopenias can have a serious impact on mother and fetus. Autoimmune thrombocytopenia (ITP) is common in pregnancies and affects both mothers and fetuses. ITP is considered to be quite benign for both groups. There has been no reported maternal mortality in more than 20 years. Significant neonatal thrombocytopenia occurs in approximately 10% and intracranial hemorrhage (ICH) in approximately 1% of cases.

NAIT is a much more serious fetal disorder. Neonatal platelet counts

The frequency of NAIT varies among ethnic groups. In caucasians, HPA-la is the most frequently implicated, HPA-5b is the second most frequently implicated.23 In rare cases a patient may develop antibodies to more than one glycoprotein. Kuijpers reported the detection of anti-HPA-lb and anti-HPA-2a alloantibodies in the maternal serum of a neonate who was severely damaged by intracranial hemorrhages. In this patient the severity of the hemorrhage was greater than would have been expected based on the neonate's moderate thrombocytopenia. They suggested that the combination of anti-HPA-lb and anti-HPA-2a alloantibodies, directed against the platelet fibrinogen receptor and the von Willebrand receptor, respectively, induced a thrombocytopathy along with the thrombocytopenia.24

The frequency of neonatal alloimmune thrombocytopenia is 1.5/ 1,000 live born neonates."25

The HPA-2b alloantigen system that is localized on the N-terminal region of glycoprotein Ib usually causes antibody formation in polytransfused patients but is only rarely implicated in NAIT.26

In Japan the frequency of NAIT is estimated at 1:3300 births. Incompatibility for HPA-4 is the major (80%) cause of NAIT, followed by HPA-3a (15%). HPA-5b induced NAIT is rare, despite that the antibody is found often (0.7%) in pregnant women.

About half of the cases of NAIT occur during the first pregnancy.27

In Japan the risk of neonatal alloimmune thrombocytopenia and refractoriness to platelet transfusion induced by the antigens of the HPAla, HPA-7a, and HPA-8a systems is extremely rare. However, based on gene frequencies attention must be paid to the involvement of the HPA4 and HPA-6W systems in these clinical disorders.28

Other low frequency platelet alloantigens have been implicated in NAIT These include HPA-8b (Sra), HPA-9b (Max), and Gov .29-31 Gro is low frequency antigen implicated in a case of NAIT in a child born to a Dutch mother. Simsek stated that the Gro antigen was inherited in an autosomal-codominant fashion.32 The antigen was localized on the glycoprotein IIb-IIIa (aIIb-b3) complex. It is estimated that the phenotype for Gor is less than 0.01% in the Dutch population.32

POST-TRANSFUSION (ISOIMMUNE) PURPURA

Post-transfusion (isoimmune) purpura (PTP) and refractory to platelet transfusion are rare conditions that are associated with the development of antibodies to the platelet specific membrane glycoproteins and to major histocompatibility complexes (HLA) antigens. HLA-DR3 is a histocompatibility phenotype that is strongly associated with alloimmunization to the PlA1 antigen.34 PTP is characterized by a sudden, profound, and self-limited thrombocytopenia (

REFRACTORY TO PLATELET TRANSFUSION

The expression "refractory to platelet transfusion" means that the patient does not respond to transfusion therapy. In these patients, infusion of platelets does not produce a sustained increase in platelet count.' The condition of refractory to platelet transfusion is usually associated with long term administration of transfused platelets. The alloantibodies produced in about two-thirds of the cases are directed against HLA antigens. Evenson has reported that antiHPA-la and anti-HPA- lb can cause refractoriness to platelet transfusions in bone marrow transplant patients. They recommend that testing for platelet-specific antibodies be considered in all patients who are refractory to HLA-matched platelets.33

METHODS OF DETECTION

The increased use of platelet transfusion over the past 30 years has exposed a larger number of individuals to antibody stimulating antigens and the development of thrombocytopenia. The number of immune thrombocytopenia cases is also likely to increase as our population grows older. To handle the increased need for detection of platelet antibodies and platelet alloantigen typing, laboratory methods must be efficient and sensitive. Methods for alloantigen typing and antibody detection include serologic techniques, ELISA methods, complement fixation, DNA typing, Western blotting, and flow cytometry.7

The Sixth International Society of Blood Transfusion Platelet Serology Workshop recently reported the results of their study. The goals of their study included establishment of antigen-typed platelet panels, determination of the correlation between serologic and DNA typing of platelet-specific antigens, and evaluation of the proficiency of techniques for the detection of platelet-specific antibodies. They compared methods for platelet-antigen typing, serologic testing, DNA techniques, allele-specific restriction site analysis, modified antigen-capture enzyme-linked immunosorbent assay, the monoclonal antibody-specific immobilization of platelet antigens assay, a mixed passive hemagglutination assay, radioimmunoprecipitation procedures, and Western blotting. They found that concordant results were achieved by using either serologic or DNA techniques to identify platelet-specific antigens. All other platelet-specific antibody assays were comparable except for the significantly lower results found with Western blotting.36

The use of flow cytometry to detect and characterize platelet specific antibodies and to evaluate platelet function has greatly increased in the last few years. This is due in part to newer techniques and the greater availability of antibodies and probes.'* Flow cytometry is a valuable tool for large scale screening to identify HPA- la-negative persons, but it can not distinguish between heterozygosity and homogzygosity. Genotyping is the assay of choice for zygosity testing, antenatal diagnosis, and for thrombocytopenic alloimmunized patients.37

A screening of 2,300 pregnant women for the platelet antigen HPAla was conducted by flow cytometry. Of the 2,300 patients, 40 (1.7%) were negative for this antigen and were at risk of developing maternal antibodies to the HPA-la antigen. This study demonstrated the potential use of flow cytometry screening for detection of individuals at risk and for use in genetic counseling. They proved the simplicity and rapidity of flow cytometry for platelet antigen screening. Their results were comparable with the Solid phase Red Cell Adherence (SRCA) method and with CPR. However, at this time, the lack of a plentiful supply of specific antibody argues against the introduction of routine screening.38

CONCLUSION

Immune hemostatic disorders are important clinically and can be devastating to the patient. At this time there is a great deal of confusion due to the lack of a standardized nomenclature for identification of platelet-specific alloantigens as well as new methodologies and equipment. However confusion should be expected in an area where new findings are occurring every day. As clinical laboratory scientists it is our responsibility to continue learning so that we can maintain our competencies and better serve the patient.

REFERENCES

1. Bithell TC: The diagnostic approach to the bleeding disorders. In: Lee GR, Bithell TC, Foerster J, Athens JW, Lukens JN, editors. Wintrobe's clinical hematology. 9th ed. Philadelphia: Lea & Febiger; 1993. p 1306. 2. George D, Bussel JB. Neonatal thrombocytopenia. Semin Thromb Hemost 1995;21:276-93.

3. Bithell TC: Thrombocytosis. In: Lee GR, Bithell TC, Foerster J, Athens JW, Lukens JN, editors. Wintrobe's clinical hematology.9th ed. Philadelphia: Lea & Febiger; 1993. p 1390-6.

4. Harker LA, Slichter SJ. The bleeding time as a screening test for evaluation of platelet function. N Engl J Med 1972;287:155. 5. Bithell TC: Pathophysiology and classification. In: Lee GR, Bithell TC, Foerster J, Athens JW, Lukens JN, editors. Wintrobe's clinical hematology. 9th ed. Philadelphia: Lea & Febiger; 1993. Chapter 49. 6. Bussel JB. Immune thrombocytopenia in pregnancy; autoimmune and alloimmune. J Re rod Immunol 1997;37:35-61.

7. Shulman NR, Reid DM. Platelet Immunology In: Colman R, Hirsh J, Marder V, Salzman EW, editors. Hemostasis and thrombosis, basic principles and clinical practice. 3rd ed. Philadelphia: JB Lippincott Co; 1994. p 427. 8. Davis, GL. Quantitative and qualitative disorders of platelets. In: StieneMartin EA, Lotspeich-Steininger CA, Koepke JA, editors. Clinical hematology, principles, procedures, correlations. 2nd ed. Philadelphia: JB Lippincott Co; 1997. P 713-34.

9. Morel-Kopp MC, Kaplan C, Proulle V, Jallu V, and others. A three amino acid deletion in glycoprotein lIla is responsible for type I Glanzmann's thrombasthenia: importance of residues Ile325Pro326Gly327 for beta3 integrin subunit association. Blood 90 1997;(2):669-77. 10. George JN, Reimann TA: Inherited disorders of the platelet membrane: Glanzmanns thrombasthenia and Bernard Soulier disease. In: Colman RW, Hirsch J, Marder VJ, Salzman EV, editors. Hemostasis and thrombosis: basic principles and clinical practice. Philadelphia: JB Lippincott; 1982. p 496.

11. Glassman AB, Shieh WJ: Neonatal alloimmune thrombocytopenia: current considerations. Ann Clin Lab Sci 1994;24:407-11. 12. Kim HO, Jin Y, Kickler TS, and others. Gene frequencies of the five major human platelet antigens in African American, white, and Korean populations. Transfusion 1995;35:8637.

13. Newman J: Nomenclature of human platelet alloantigens: a problem with the HPA system? Blood 1994;83:1447-51. 14. von dem Borne AEGK, Decary E Nomenclature of platelet-specific antigens. Transfusion 1990;30:477.

15. von dem Borne AEGK, Decary F: ICSH/ISBT working party on platelet serology. Nomenclature of platelet-specific antigens. Vox Sang 1990;58:176-8.

16. von dem Borne AEGK, Kaplan C, Minchinton R. Nomenclature of human platelet alloantigens [letter; comment] Blood 1995;85:1409-10. 17. Schmitz G, Rothe G, RufA, and others. European working group on clinical cell analysis: Consensus protocol for flow cytometric characterization of platelet function. Thromb Haemost 1998;79:885-96. 18. Wautier, JL. Nomenclature of human platelet alloantigens [letter to the editor]. Blood 1994;84:989.

19. Jackson GM, Scott JR. Alloimmune conditions and pregnancy. Baillieres Clin Obstet Gynaecol 1992;6:541-63. 20. Beadling WV, Herman JH, Stuart MJ, and others. Fetal bleeding in neonatal alloimmune thrombocytopenia mediated by anti-PEAl is not associated with inhibition of fibrinogen binding to platelet GPIIb/IIIa. Am J Clin Pathol 1995;103:636-41.

21. Kunicki TJ, Newman J. The molecular immunology of human platelet proteins. Blood 1992;80:1386-404.

22. Bussel JB. Immune thrombocytopenia in pregnancy: autoimmune and alloimmune. J Reprod Immunol 1997;37:35-61. 23. Kalb R, Santoso S, Kiefel V, Mueller-Eckhardt C. Molecular biologic clarification of Br alloantigens in human platelets and its application in DNA typing, Beitr Infusionsther Transfusionsmed 1994;32:217-20. 24. Kuijpers RW, van den Anker JN, Baerts W, von dem Borne AE. A case of severe neonatal thrombocytopenia with schizencephaly associated with anti-HPA-lb and anti-HPA-2a. BrJ Haematol 1994;87:576-9. 25. Dreyfus M, Kaplan C, Verdy E, and others. Frequency of immune thrombocytopenia in newborns: a prospective study. Immune Thromboc,vtopenia Working Group. Blood 1997;89:4402-6.

26. Kroll H, Muntean W, Kiefel V, and others. Anti Ko(a) as a cause of neonatal alloimmune thrombocytopenia, Beitr Infusionsther Transfusionsmed 1994;32:244-6.

27. Ohto H. Neonatal alloimmune thrombocytopenia. Nippon Rinsho 1997;55:2310-4.

28. Tanaka S, Ohnoki S, Shibata H, and others. Gene frequencies of human platelet antigens on glycoprotein IIIa in Japanese. Transfusion 1996;36(9):813-7.

29. Santoso S, Kalb R, Kroll H, and others. A point mutation leads to an unpaired cysteine residue and a molecular weight polymorphism of a functional platelet beta 3 integrin subunit. The Sra alloantigen system of GPIIIa. J Biol Chem 1994;269:8439-44.

30. Norris P, Simsek S, de Bruijne-Admiraal LG, Porcelijn L, and others. Max(a), a new low-frequency platelet-specific antigen localized on glycoprotein Ilb, is associated with neonatal alloimmune thrombocytopenia. Blood 1995;86(3):1019-26.

31. Bordin JO, Kelton JG, Warner MN, and others. Maternal immunization to Gov system alloantigens on human platelets. Transfusion 1997;37:823-8.

32. Simsek S, Vlekke AB, Kuijpers RW, and others. A new private platelet antigen, Groa, localized on glycoprotein IIIa, involved in neonatal alloimmune thrombocytopenia. Vox Sang 1994;67:302-6. 33. Evenson DA, Stroncek DF, Pulkrabek S, and others. Posttransfusion putpura following bone marrow transplantation. Transfusion 1995;35:688-93. 34. Blanchette VS, Chen L, de Friedberg ZS, and others. Alloimmunization to the PIA 1 platelet antigen: results of a prospective study. Br J Haematol 1990;74:209-15.

35. Lucas GF, Pittman SJ, Davies S, and others. Post-transfusion purpura (PTP) associated with anti-HPA-la, anti-HPA-2b and anti-HPA-3a antibodies. Transfus Med 1997;7:295-9.

36. Teramura G, Slichter SJ. Report on the Sixth International Society of Blood Transfusion Platelet Serology Workshop. Transfusion 1996;36:75-81. 37. Tazzari PL, Cirillo D, Bontadini A, and others. Flow cytometry immunophenotyping and polymerase chain reaction-site-specific primers genotyping for HPA-1 alloantigens in an Italian blood donor population. Vox Sang 1998;74:42-5.

38. Lavu EK, Nelson M, Po HJ, and others. Antenatal screening for HPAla by flow cytometry. Aust N Z J Obstet Gynaecol 1997;37:180-3.

Gerald L Davis PhD is Professor of Medical Technology and Physiol ogy at Michigan State University, East Lansing, Ml.

Address for correspondence: Gerald L Davis PhD, Medical Technol ogy Program, 322 North Kedzie Lab, Michigan State University East Lansing, Michigan 48823. (517) 353-7800, (517) 432-2006(fax).

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