Richard H. Aster

Antibodies from patients with heparin-induced thrombocytopenia/thrombosis are specific for platelet factor 4 complexed with heparin or bound to endothelial cells.

Heparin-induced thrombocytopenia/thrombosis (HITP) is thought to be mediated by immunoglobulins that activate platelets in the presence of pharmacologic concentrations of heparin, but the molecular basis for this relatively common and often serious complication of heparin therapy has not been established. We found that plasma from each of 12 patients with HITP contained high titer (> or = 1:200) antibodies that reacted with immobilized complexes of heparin and platelet factor 4 (PF4), a heparin-binding protein contained in platelet alpha-granules. Recombinant human PF4 behaved similarly to PF4 isolated from platelets in this assay system. Complexes formed at an apparent heparin/PF4 molecular ratio of approximately 1:2 (fresh heparin) and approximately 1:12 (outdated heparin) were most effective in binding antibody. Immune complexes consisting of PF4, heparin, and antibody reacted with resting platelets; this interaction was inhibited by a monoclonal antibody specific for the Fc gamma RII receptor and by excess heparin. Human umbilical vein endothelial cells, known to express heparin-like glycosaminoglycan molecules on their surface, were recognized by antibody in the presence of PF4 alone; this reaction was inhibited by excess heparin, but not by anti-Fc gamma RII. Antibodies reactive with heparin/PF4 were not found in normal plasma, but IgG and IgM antibodies were detected at dilutions of 1:10 (IgG) and 1:50 (IgM) in 3 of 50 patients (6%) with other types of immune thrombocytopenia. These findings indicate that antibodies associated with HITP react with PF4 complexed with heparin in solution or with glycosaminoglycan molecules on the surface of endothelial cells and provide the basis for a new hypothesis to explain the development of thrombocytopenia with thrombosis or disseminated intravascular coagulation in patients sensitive to heparin.

The human platelet alloantigens, PlA1 and PlA2, are associated with a leucine33/proline33 amino acid polymorphism in membrane glycoprotein IIIa, and are distinguishable by DNA typing.

The human platelet alloantigens, PlA1 and PlA2, comprise a diallelic antigen system located on a component of the platelet fibrinogen receptor, membrane glycoprotein (GP) IIIa. Of the known platelet alloantigens, PlA1, which is carried by 98% of the caucasian population, appears to be the alloantigen that most often provokes neonatal alloimmune thrombocytopenic purpura and posttransfusion purpura. The structural features of the GPIIIa molecule responsible for its antigenicity are as yet unknown. Using the polymerase chain reaction (PcR), we amplified the NH2-terminal region of platelet GPIIIa mRNA derived from PlA1 and PlA2 homozygous individuals. Nucleotide sequence analysis of selected amplified cDNA products revealed a C in equilibrium T polymorphism at base 196 that created a unique Nci I restriction enzyme cleavage site in the PlA2, but not the PlA1 form of GPIIIa cDNA. Subsequent restriction enzyme analysis of cDNAs generated by PcR from 10 PlA1/A1, 5 PlA2/A2, and 3 PlA1/A2 individuals showed that Nci I digestion permitted clear discrimination between the PlA1 and PlA2 alleles of GPIIIa. All PlA2/A2 individuals studied contain a C at base 196, whereas PlA1 homozygotes have a T at this position. This single base change results in a leucine/proline polymorphism at amino acid 33 from the NH2-terminus, and is likely to impart significant differences in the secondary structures of these two allelic forms of the GPIIIa molecule. The ability to perform DNA-typing analysis for PlA phenotype may have a number of useful clinical applications, including fetal testing and determination of the phenotype of severely thrombocytopenic individuals.

A Study of Variables Affecting the Quality of Platelets Stored at “Room Temperature”

The effect of variables associated with the donor and with methods of collecting, processing, and storing platelets on the quality of platelets kept at ambient temperature was studied. Changes in structural integrity of platelets, decrease in pH, loss of aggregability, and kinetics in vivo of platelets tagged with 51Cr were used as indicators of the tolerance of platelets to storage. A platelet concentration of less than 2.5 × 106 per cu mm, a temperature of storage less than 24 C, and continuous, gentle, agitation were found to be essential for satisfactory preservation of platelet integrity, function, and posttransfusion survival. Platelets from female donors tolerated storage less well than did platelets from male donors, possibly because the lower hematocrit of blood collection from females resulted in greater initial acidity of the concentrate. A number of other variables analyzed appear to be of little or no consequence for successful platelet storage.

Heparin-Induced Thrombocytopenia and Thrombosis

&NA; Thrombocytopenia and thrombosis, recognized complications of heparin therapy, have long been thought to be antibody mediated. However, in vitro studies have failed to provide satisfactory explanations for platelet destruction and paradoxical thrombosis in patients with heparin sensitivity. Recently, several groups of investigators have shown that plasma from patients with heparin-induced thrombocytopenia and thrombosis contains IgG and IgM antibodies specific for complexes containing heparin and platelet factor 4, a heparin-binding protein normally contained in platelet alpha granules. These observations have provided new insights into the pathogenesis of this serious side-effect of heparin therapy and should point the way to improved diagnosis, prevention, and, possibly, treatment.

Patients treated with unfractionated heparin during open heart surgery are at high risk to form antibodies reactive with heparin:platelet factor 4 complexes.

Recent studies have demonstrated a strong association between type II (immunologically mediated) heparin-induced thrombocytopenia/thrombosis (HITP) and antibodies reactive with complexes consisting of heparin and platelet factor 4 (PF4), a heparin-binding protein normally found in platelet-alpha granules. However, the frequency with which such antibodies develop in patients given treatment with heparin has not yet been defined. We studied the development of heparin:PF4-specific antibodies in 51 patients who received a single dose of unfractionated heparin (UFH) during cardiac catheterization and were then given UFH or low-molecular-weight heparin (LMWH) again during and after open heart surgery. Eleven of the 51 patients (22%) had antibodies reactive with heparin:PF4 when they were admitted for cardiac surgery; these antibodies were mainly of the immunoglobulin M (IgM) class and were apparently stimulated by exposure to UFH at cardiac catheterization. Seventeen of 34 patients (50%) without preexisting antibody who were given UFH during and for 1 to 3 days after surgery formed immunoglobulin G antibodies or IgM antibodies (or both) by the sixth postoperative day. Overall, 27 of 44 patients (61%) who were given UFH at surgery had antibodies by the time of hospital discharge. None of 6 patients without preexisting antibody who were given LMWH at surgery formed antibodies (p < 0.03). However, LMWH was given as a single injection only on the day of surgery. The titer of the antibodies formed by patients receiving UFH ranged from 1:10 to 1:200, significantly lower than those in patients with a clinical diagnosis of HITP. Moderate thrombocytopenia was common after open heart surgery, but platelet levels in patients who had preexisting antibodies or formed new antibodies did not differ significantly from those in patients without antibody. Clinically significant thrombosis did not develop in any patient and HITP was not diagnosed in any patient. Antibodies reactive with heparin:PF4 formed in only 3 of 66 patients (4.5%) undergoing other types of surgery. One of these patients had been given UFH 3 months previously; the other 2 may have been exposed to heparin used to flush intravenous lines postoperatively. No antibodies reactive with heparin:PF4 were found in any of 108 normal subjects. We conclude that UFH is more immunogenic than has been thought and that patients exposed to this anticoagulant during open heart surgery are at high risk to form low titer (</= 1:200) antibodies reactive with heparin:PF4. Further studies are needed to determine whether such antibodies are clinically significant--that is, whether sensitized patients are at risk to develop HITP if heparin treatment is continued for more than 1 to 3 days or is reinstituted at a later date.

TRALI due to granulocyte‐agglutinating human neutrophil antigen‐3a (5b) alloantibodies in donor plasma: a report of 2 fatalities

BACKGROUND : TRALI is usually an immunologic reaction to WBC antibodies in infused plasma and ranks second only to ABO mismatch as a cause of transfusion‐associated death. Implicated donors are usually multiparous women (≥3 pregnancies).

Drug-induced immune thrombocytopenia: pathogenesis, diagnosis, and management.

Summary.  Drug‐induced immune thrombocytopenia (DITP) can be triggered by a wide range of medications. Although many cases of DITP are mild, some are characterized by life‐threatening bleeding symptoms. The pathogenesis of DITP is complex, in that at least six different mechanisms have been proposed by which drug‐induced antibodies can promote platelet destruction. It is possible in many cases to identify antibodies that react with platelets in the presence of the sensitizing drug, but the required testing is technically demanding and not widely available. Therefore, a decision on whether to discontinue an implicated medication in a patient suspected of having DITP must be made on clinical grounds. An algorithm is available that can be helpful in assessing the likelihood that a particular drug caused thrombocytopenia, but the most important aspects of patient management are a high index of suspicion and a careful history of drug exposure in an individual who presents with acute, often severe thrombocytopenia of unknown etiology. How drugs induce platelet‐reactive antibodies and how, once formed, the antibodies cause platelet destruction following exposure to the drug is poorly understood. Further studies to address these issues and characterize more completely the range of drugs and drug metabolites that can cause DITP are needed.

Enzymatic amplification of platelet-specific messenger RNA using the polymerase chain reaction.

Human platelets are derived from megakaryocytes as anucleate cells, and thus contain only vestigial amounts of RNA capable of being transcribed into protein. This has greatly hampered efforts to study directly platelet-specific gene products and their associated polymorphisms. In this report, we describe direct amplification, using the polymerase chain reaction, of platelet-derived mRNA in amounts sufficient to permit detailed analysis, such as restriction mapping and nucleotide sequencing. The ability to generate large amounts of cDNA from platelet-specific mRNA sequences should make possible direct molecular characterization of normal platelet proteins, and facilitate the investigation of a wide variety of inherited platelet disorders.

Human platelet antigen-specific alloantibodies implicated in 1162 cases of neonatal alloimmune thrombocytopenia

BACKGROUND:  Neonatal alloimmune thrombocytopenia (NATP) caused by fetomaternal mismatch for human platelet (PLT) alloantigens (HPAs) complicates approximately 1 in 1000 to 1 in 2000 pregnancies and can lead to a serious bleeding diathesis, intracranial hemorrhage, and sometimes death of the fetus or neonate. As a national reference center for NATP investigations, our experience with this entity over a 12‐year period was reviewed.

Studies of Platelet Concentrates Stored at 22 C and 4 C

As measured by stability of pH, aggregability, and structure, concentrated platelets are better preserved for 72 hours at 4 C than at 22 C. While survival in vivo, as determined with 51Cr, of platelets stored at room temperature is extremely variable, survival of platelets stored at 4 C is invariably shortened to two to three days. In the treatment of bleeding in thrombocytopenic patients, however, platelets kept at 4 C for 24 to 72 hours are superior to those kept at 22 C with respect to their capacity to shorten the bleeding time and effect hemostasis. The relative ineffectiveness of platelets kept at 22 C for 72 hours appears to be the result of a functional defect which develops during storage at ambient temperature.