The Scripps Research Institute

Scripps Clinic and Research Foundation

Robert McMillan

Mark H Ginsberg

Edward F Plow

Ronald D Bowditch

Polypeptides and fusion polypeptides that immunologically mimic the native Pl.sup.A1 and Pl.sup.A2 determinants of platelet protein GPIIIa are disclosed. Also disclosed are DNA segments and recombinant DNA molecules that encode those polypeptides and fusion polypeptides and methods for expressing and using those determinants. Antibodies that immunoreact with one determinant or the other, but not both are also disclosed, as are methods of preparing and using those antibodies.

Platelet GPIII P1.sup.A1 and P1.sup.A2 epitopes, their preparation and

The present invention discloses polypeptides consisting essentially of an amino acid residue sequence corresponding to a formula selected from the group consisting of: Tyr-His-Asp-Arg-Lys-Glu-Phe-Ala-Lys-Phe-Glu-Glu-Glu-Arg-Ala-Arg-Ala-Lys-Tr p-Asp-Thr-Ala-Asn-Asn (SEQ ID NO 1); Ala-Asn-Asn-Pro-Leu-Tyr-Lys-Glu-Ala-Thr-Ser-Thr-Phe-Thr-Asn-Ile-Thr-Tyr-Ar g-Gly-Thr (SEQ ID NO 2); Ile-His-Asp-Arg-Lys-Glu-Phe-Ala-Lys-Phe-Glu-Glu-Glu-Arg-Ala-Arg-Ala-Lys-Tr p-Asp-Thr-Ala-Asn-Asn-Pro-Leu-Tyr-Lys-Glu-Ala-Thr-Ser-Thr-Phe-Thr-Asn-Ile-T hr-Tyr-Arg-Gly-Thr (SEQ ID NO 4); and Gly-Pro-Asp-Ile-Leu-Val-Val-Leu-Leu-Ser-Val-Met-Gly-Ala-Ile-Leu-Leu-Thr-Gl y-Leu-Ala-Ala-Leu-Leu-Ile-Trp-Lys-Leu-Leu-Ile-Thr-Ile-His-Asp-Arg-Lys-Glu-P he-Ala-Lys-Phe-Glu-Glu-Glu-Arg-Ala-Arg-Ala-Lys-Trp-Asp-Thr-Ala-Asn-Asn-Pro- Leu-Tyr-Lys-Glu-Ala-Thr-Ser-Thr-Phe-Thr-Asn-Ile-Thr-Tyr-Arg-Gly-Thr (SEQ ID NO 5), as well as diagnostic kits including same. The present invention also discloses methods for determining the presence of anti-GPIIIa autoantibodies in a vascular fluid sample, which autoantibodies are indicative of chronic immune thrombocytopenic purpura in a patient.

Methods and compositions for diagnosing chronic immune thrombocytopenic

A polypeptide consisting essentially of an amino acid residue sequence corresponding to a formula selected from the group consisting of:

The present invention provides a method for assaying a sample of cells for the presence of a cell surface antigen. More particularly, the present invention describes methods for detecting the presence of plasma and cell bound autoantibodies against cell surface antigens. In addition, the present invention provides a method of crossmatching donor platelets and transfusion recipients.

Cell surface antigen detection method

A polypeptide consisting essentially of an amino acid residue sequence selected from the group consisting of:

Chronic immune thrombocytopenic purpura is due to platelet destruction by circulating anti-platelet antibody. Although autoantibodies against the platelet glycoprotein IIb/IIIa complex and glycoprotein Ib have been demonstrated using various methods, practical assays for detection of platelet-associated or plasma autoantibodies have not been available. I studied 44 patients with chronic immune thrombocytopenic purpura where platelet-associated and plasma autoantibodies against the glycoprotein IIb/IIIa complex and glycoprotein Ib were measured using a newly developed immunobead assay and a previously reported microtiter well assay. Platelet-associated autoantibody was detected using the immunobead assay in nine of 11 patients (81.8%; seven with anti-GPIIb/IIIa, two with anti-GPIb). Plasma autoantibodies were noted in 28 of 44 patients (63.6%; 19 with anti-GPIIb/IIIa, seven with anti-GPIb and two with both). Positive results were noted in 24 of 44 patients using the immunobead assay and in only 12 of 44 using the microtiter well assay, suggesting that solubilization of the platelets prior to antibody addition, as in the microtiter well assay, alters epitope stability. Of the 20 thrombocytopenic control patients studied, all gave negative results using both assays. I conclude that these assays allow detection of autoantibodies in the majority of patients with chronic immune thrombocytopenic purpura confirming the presence of an autoimmune process. In addition, the two assays may differentiate between epitope types.

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Del Mar, CA, United States of America

Robert McMillan