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Heparin-induced thrombocytopenia (HIT) is a thrombotic complication of heparin therapy. The most used functional method for HIT diagnosis is serotonin release assay (SRA). A different functional method based on ATP release with luciferin/luciferase long-life and stable luminescent signal is used here, which is shown to be comparable for accuracy with SRA in both negative (patients 4Ts ≤3, and negative for both anti-PF4/heparin immunoassay and SRA) and positive (4Ts >3, and positive for both PF4/heparin antibodies and SRA) patients. Our results show that ATP release is higher in washed platelets activated by sera from positive patients than in platelets activated by sera from negative patients. In conclusion, we demonstrate that ATP release assay is a valid alternative method to SRA for the identification of pathogenic anti-PF4/heparin antibodies.

Recent studies have shown that antibodies characteristic of quinine- and quinidine-induced thrombocytopenia sometimes recognize the platelet membrane glycoprotein (GP) complex IIb/IIIa in addition to their well known target, GPIb/IX. We have investigated the frequency with which drug-induced antibodies bind to GPIIb/IIIa and the nature of their target epitopes. In studies of sera from 13 patients sensitive to quinidine or quinine, we found that 10 contained IgG antibodies specific for both GPIb/IX and GPIIb/IIIa, two reacted with GPIb/IX alone, and one reacted with GPIIb/IIIa alone. In all cases, the presence of drug was required for binding of IgG to target GPs. By immunoabsorption, we found that each of five polyspecific sera contained at least two different antibodies, one reactive with GPb/IX and the other with GPIIb/IIIa. Further studies with eight drug- dependent antibodies (DDAb) specific for GPIIb/IIIa showed that three recognized the GPIIb/IIIa complex only, one recognized GPIIb alone, and three recognized GPIIIa alone. The eighth serum appeared to bind to both GPIIIa alone and to an epitope determined by the GPIIb/IIIa complex. The three antibodies specific for GPIIIa alone also reacted with GPIIIa deglycosylated with endo-H, and with the major (61 Kd) fragment obtained by chymotryptic digestion of GPIIIa but failed to react with reduced GPIIIa. These findings demonstrate that, in drug- induced, immunologic thrombocytopenia, the anti-platelet immune response is typically directed against epitopes on both GPIb/IX and GPIIb/IIIa. The three DDAb we studied that were specific for GPIIIa alone recognize epitopes resistant to chymotrypsin and endo-H treatment that are dependent on intrachain disulfide bonding.

Human PF4 is a heparin-binding chemokine known to be capable of inhibiting endothelial cell proliferation and angiogenesis. To explore the biological mechanisms responsible for this action, we investigated the effect of PF4 on epidermal growth factor (EGF)-stimulated human umbilical vein endothelial cells (HUVEC), a model system in which stimulation is essentially independent of interaction with cell-surface glycosaminoglycans. Based on previous findings that PF4 blocks endothelial cell cycle entry and progression into S phase, we studied the molecular mechanism(s) of PF4 interference with cell cycle machinery. PF4 treatment of EGF-stimulated HUVEC caused a decrease in cyclin E-cyclin-dependent kinase 2 (cdk2) activity with resulting attenuation of retinoblastoma protein phosphorylation. PF4-dependent downregulation of cyclin E-cdk2 activity was associated with increased binding of the cyclin-dependent kinase inhibitor, p21(Cip1/WAF1), to the cyclin E-cdk2 complex. Analysis of total cellular p21(Cip1/WAF1) showed that in the presence of PF4, p21(Cip1/WAF1) levels were sustained at time points when p21(Cip1/WAF1) was no longer detectable in cells stimulated by EGF in the absence of PF4. These findings indicate that PF4 inhibition of HUVEC proliferation in response to EGF is associated with impaired downregulation of p21(Cip1/WAF1) and provide the first evidence for interference with cell cycle mechanisms by a chemokine.

The IIb-IIIa glycoprotein complex is a favored target for allo-, auto-, and drug-dependent antibodies associated with immune thrombocytopenia. A soluble, recombinant form of the GPIIb-IIIa heterodimer that could be produced in large quantities and maintained in solution without detergent could provide a useful experimental tool for the study of platelet-reactive antibodies, but previous attempts to produce such a construct have yielded only small quantities of the end product. Using a baculovirus expression system and the dual-promoter transfer vector P2Bac, we were able to express soluble GPIIb-IIIa complex (srGPIIb-IIIa) lacking cytoplasmic and transmembrane domains in quantities of about 1,000 microg/L, about 40 times greater than reported previously. The high yield achieved may be related to inclusion of the entire extracellular region of the GPIIb light chain in the construct. srGPIIb-IIIa reacts spontaneously with fibrinogen, and this interaction is totally inhibited by the peptide RGDS. Reactions of 24 GPIIb-IIIa-specific antibodies evaluated (12 monoclonal, 3 allo-specific, 3 auto-specific, and 6 drug-dependent) with srGPIIb-IIIa were indistinguishable from reactions with platelet GPIIb-IIIa. Thus, srGPIIb-IIIa spontaneously assumes an active, ligand-binding conformation and contains epitopes for all monoclonal and human antibodies tested to date. srGPIIb-IIIa can be produced in large quantities, can readily be modified by site-directed mutagenesis, and should facilitate identification of epitopes recognized by GPIIb-IIIa-specific antibodies, study of the mechanism(s) by which certain drugs promote antibody binding to GPIIb-IIIa in drug-induced thrombocytopenia and structure-function relationships of GPIIb-IIIa.

Previous studies have suggested that PECAM-1 mediates cellular interactions via both homophilic and heterophilic adhesive mechanisms. Cell surface glycoaminoglycans have been implicated as one of the heterophilic ligands for PECAM-1. To determine whether PECAM-1 is capable of interacting directly with glycosaminoglycans, we examined the adhesive properties of multiple monovalent and multivalent forms of this adhesion molecule. We found that the binding of a bivalent PECAM-1/IgG chimeric protein or multivalent PECAM-1-containing proteoliposomes to multiple different cell lines was 1) strictly dependent upon cell surface expression of PECAM-1 and 2) unaffected by the presence of excess heparin or heparan sulfate. The extracellular domain of PECAM-1 failed to interact specifically with heparin-Sepharose, 3H-labeled heparin, or a heparin-bovine serum albumin conjugate. In addition, an amino acid sequence motif inadvertently created by the juxtaposition of PECAM-1 and IgG sequences within the hinge region of certain PECAM-1/IgG chimeric constructs was found to confer glycosaminoglycan binding properties not normally present within the extracellular domain of the native molecule. Together, these data suggest that the mechanism by which heparin is able to affect PECAM-1-dependent cell-cell adhesion is indirect and occurs via inhibition of events that occur downstream from PECAM-1 engagement.

Recent reports indicate that antibodies associated with heparin-induced thrombocytopenia and thrombosis (HITP) are specific for complexes formed between heparin and the heparin-binding, platelet alpha granule protein, platelet factor 4 (PF4). As with other disorders mediated by immune complexes (IC), the characteristics of the involved immunoglobulins could affect the ability of IC to cause symptoms. We therefore studied the class, subclass, and potency of antibodies specific for heparin:PF4 complexes formed by two groups of patients: one with severe thrombocytopenia, with or without thrombosis, and a positive serotonin release assay (SRA) (Group 1) and another with mild or absent thrombocytopenia, absence of thrombosis, and a negative SRA despite having formed antibodies reactive with heparin:PF4 complexes (Group 2). IgG antibodies were more common in the Group 1 patients (100%) than in Group 2 (46%), whereas IgM antibodies were more common in Group 2 (81%) than in Group 1 (42%) (P = 0.009). About half of each group formed IgA antibodies. In each group, the IgG antibodies were predominantly IgG1 (82%); 42% were IgG3. Only one IgG2 antibody was identified in a total of 52 antibody formers. Antibodies of the IgG class were consistently of higher titer in Group 1 patients than in Group 2 patients (P0.001). Recent reports suggest that the H131 form of the Fc gammaRII receptor, which binds preferentially to IgG2 Fc, is found with greater than expected frequency in patients with HITP. Identification of only one IgG2 antibody among 38 antibodies of the IgG class argues against a unique role for antibodies of this subclass in the pathogenesis of HITP. The finding that titers of antibodies in Group 1 patients were a significantly higher titer than in Group 2 patients suggests that development of the full-blown HITP syndrome may require the formation of antibodies of unusually high titer.

To assess the potential contribution of the cysteine-rich domain of human platelet glycoprotein (GP) IIIa to the structure of the PlA1 epitope, we used site-directed mutagenesis to substitute alanines for cysteines at key positions potentially involved in PlA1 alloantigen formation. One of these GPIIIa isoforms in which alanine replaced Cys435 reacted normally with a series of well-characterized murine MoAbs directed against the 250 amino-terminal residues of GPIIIa, whereas binding of MoAbs specific for residues 348-692 was diminished or lost. Interestingly, of eight PlA1-specific antibodies tested, two recognized Ala435GPIIIa, two did not, and four bound to it less well than to wild-type (WT) GPIIIa. However, disruption of disulfide bonds located at or near the N-terminus of GPIIIa abolished the binding of all the anti-PlA1 alloantibodies tested. These findings provide evidence that the humoral response to the PlA1 antigen is (1) heterogenous, ie, the binding site on GPIIIa for human anti-PlA1 antibodies differs from one individual to another and (2) complex--although some anti-PlA1 alloantibodies bind to an epitope comprised solely of the amino-terminus of GPIIIa, others combine most efficiently with a more complex determinant that involves the cysteine-rich domain of GPIIIa. These findings may have implications for diagnostic and therapeutic use of synthetic or recombinant PlA1 mimetics.

The human Baka/Bakb alloantigen system has been implicated in the pathogenesis of post-transfusion purpura and neonatal alloimmune thrombocytopenic purpura. Human alloantisera specific for either the Baka or Bakb allele have been shown to react exclusively with the heavy chain of membrane glycoprotein (GP) IIb. To investigate the structure of the Bak epitopes, we used the polymerase chain reaction (PCR) to amplify GPIIb cDNA synthesized from platelet RNA samples prepared from individuals of known serologic phenotype. Subsequent DNA sequence analysis of amplified GPIIb cDNAs derived from one Baka homozygous individual and one Bakb homozygous individual revealed a single nucleotide base difference near the 3' end of the mRNA encoding the GPIIb heavy chain. Short 13 base allele-specific oligonucleotides (ASO) containing the putative phenotype-specific base in the middle were then synthesized, end-labeled with digoxigenin-11-dUTP using terminal transferase, and used as probes in subsequent dot-blot hybridization experiments. Platelet RNA was prepared from a panel made up of four Baka/a, three Bakb/b, and two Baka/b individuals, and the mRNA encoding GPIIb was amplified using PCR and spotted onto nylon membranes. ASO hybridization showed that the nucleotide base difference identified above segregated with Bak phenotype in all nine individuals examined (P = .002). The base pair substitution results in an amino acid polymorphism at residue 843 of the mature heavy chain. The Baka form of GPIIb encodes an isoleucine at this position, whereas the Bakb allele contains a serine. Identification of the polymorphism associated with this clinically important alloantigen system should permit new therapeutic and diagnostic approaches for treating and managing patients with alloimmune thrombocytopenic disorders.