This report describes studies into the pathophysiology of heparin- induced thrombocytopenia. The IgG fraction from each of nine patients with heparin-induced thrombocytopenia caused heparin-dependent platelet release of radiolabeled serotonin. Both the Fc and the Fab portions of the IgG molecule were required for the platelet reactivity. The platelet release reaction could be inhibited by the Fc portion of normal human or goat IgG, and patient F(ab')2, but not F(ab')2 from healthy controls. These results suggested that the Fab portion of IgG binds to heparin forming an immune complex and the immune complexes initiate the platelet release reaction by binding to the platelet Fc receptors. To directly challenge this hypothesis, we preincubated the serotonin-labeled platelets with the monoclonal antibody against the platelet Fc receptor (IV.3). This monoclonal antibody completely inhibited the release reaction caused by heparin and patient sera, as well as heat aggregated IgG, but did not block collagen or thrombin- induced platelet release. Heparin-dependent platelet release also could be inhibited in vitro by the addition of monocytes and neutrophils, but not by red cells, presumably because the Fc receptors on the phagocytic cells have a higher binding affinity for IgG complexes than do platelets. Platelets from patients with congenital deficiencies of specific glycoproteins Ib and IX (Bernard-Soulier syndrome) and IIb and IIIa (Glanzmann's thrombasthenia) displayed normal heparin-dependent release indicating that the release reaction did not require the participation of these glycoproteins. These studies indicate that heparin-induced thrombocytopenia is an IgG-heparin immune complex disorder involving both the Fab and Fc portion of the IgG molecule.
Heparin-induced thrombocytopenia (HIT) is an important complication of heparin therapy. Although there is general agreement that platelet activation in vitro by the HIT IgG is mediated by the platelet Fc receptor, the interaction among the antibody, heparin, and platelet membrane components is uncertain and debated. In this report, we describe studies designed to address these interactions. We found, as others have noted, that a variety of other sulfated polysaccharides could substitute for heparin in the reaction. Using polysaccharides selected for both size and charge, we found that reactivity depended on two independent factors: a certain minimum degree of sulfation per saccharide unit and a certain minimum size. Hence, highly sulfated but small (< 1,000 daltons) polysaccharides were not reactive nor were large but poorly sulfated polysaccharides. The ability of HIT IgG to recognize heparin by itself was tested by Ouchterlony gel diffusion, ammonium sulfate and polyethylene glycol precipitation, and equilibrium dialysis. No technique demonstrated reactivity. However, when platelet releasate was added to heparin and HIT IgG, a 50-fold increase in binding of radio-labeled heparin to HIT IgG was observed. The releasate was then depleted of proteins capable of binding to heparin by immunoaffinity chromatography. Only platelet factor 4-immunodepleted releasate lost its reactivity with HIT IgG and heparin. Finally, to determine whether the reaction occurred on the surface of platelets or in the fluid phase, washed platelets were incubated with HIT IgG or heparin and after a wash step, heparin or HIT IgG was added, respectively. Reactivity was only noted when platelets were preincubated with heparin. Consistent with these observations was the demonstration of the presence of PF4 on platelets using flow cytometry. These studies indicate that heparin and other large, highly sulfated polysaccharides bind to PF4 to form a reactive antigen on the platelet surface. HIT IgG then binds to this complex with activation of platelets through the platelet Fc receptors.
The Gova/b alloantigens are expressed on a 175-kD protein (GP175) on human platelets. Anti-Gov alloantibodies have been implicated in posttransfusion purpura and alloimmune neonatal thrombocytopenia. In this report we characterize the immunochemistry of the alloantigens and identify the platelet protein that expresses the Gov epitopes. Approximately 50% of GP175 containing the Gov epitope was released from platelets treated with phosphatidylinositol-specific phospholipase C, indicating that at least some of this protein exists as a glycosylphosphatidylinositol (GPI)-linked isoform. Radioimmunoprecipitation and immunodepletion studies indicated that the Gova/b alloantigens are expressed on the GPI-anchored CDw109 protein. The Gova/b epitopes were expressed on an extracellular, 120-kD soluble fragment (p120) of CDw109 produced by calcium-dependent protease cleavage. Anti-Gov immunoprecipitates of chymotryptic digests of p120 contained 70- and 52-kD fragments of CDw109. Deglycosylation of native CDw109 had no effect on recognition by Gov alloantisera; however, the epitopes were destroyed after exposure to sodium dodecyl sulfate. Gova/b alloantigens were expressed on platelets and PHA-activated T-cells, cultured human umbilical vein endothelial cells, and by many different tumor cell lines, consistent with the tissue distribution of CDw109.
In this report we describe a platelet alloantigen system that is carried on a novel platelet protein of 175 Kd. Antisera against the two alleles (Gova/Govb) were found in two patients who had received large numbers of platelet transfusions. The anti-Gov alloantibodies could not be detected using a whole platelet solid phase enzyme immunoassay, or by a platelet glycoprotein capture enzyme immunoassay using monoclonal antibodies against glycoproteins Ib/IX, Ia/IIa, and IIb/IIIa. Using radioimmunoprecipitation techniques, a protein was precipitated that migrated at 175 Kd (reduced). Under nonreduced conditions, a 150-Kd protein was detected with a minor component at 175Kd. The detection of the alloantigens was not activation-dependent. Using immunodepletion studies, we demonstrated that each alloantiserum recognized an epitope on a discrete population of the 175-Kd platelet protein. Family studies demonstrated that the alloantigens designated as Gova and Govb were inherited in an autosomal codominant fashion. The phenotypic frequencies were Gova/Gova, 26%; Gova/Govb, 55%; Govb/Govb, 19%; giving gene frequencies of 0.532 and 0.468 for Gova and Govb, respectively (n = 33).
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