The immunogenicity of Gov alloantigens is comparable to that of the HPA-5 system

Severe thrombocytopenia of the newborn is most often caused by alloantibodies to platelet antigens (a-HPA 1a).
The presence of alloantibodies may be suspected in newborns with signs or symptoms of thrombocytopenia. When a-HPA 1a is detected in a pregnant woman, there is no general agreement on follow up in pregnancy or about time and mode of delivery. The aim of the present study was to identify pregnancies at risk, follow the women with a-HPA 1a antibodies determinations and clinical evaluations and plan the delivery as safe as possible with respect to severe bleedings.
R esults The frequency of HPA 1bb was found to be 2·1% (n=17,000) and in 12·0% of the HPA 1bb women, anti-HPA 1a antibodies were detected. Fourteen babies were born with thrombocytopenia of < 35 × 10⁹ L⁻¹ and transfused. There was a clear correlation between antibody level in the mother and the severity of thrombocytopenia in the newborns. Beside from one clinical insignificant intracranial bleeding (ICH) detected by CT, all the children were healthy.
D iscussion Based on earlier publications, 5–6 babies in our material should have had ICH with neurological sequels. We believe that the combination of early detection of antibodies, close clinical follow up, termination of the pregnancy at week 38 by Caesarean section and immediate compatible platelet transfusion may explain the profitable clinical outcome for the babies. This relatively simple regimen is beneficial for improvement of perinatal morbidity and mortality.     

Effect of methylprednisolone on human lymphocytes primed to alloantigens

Human lymphocytes were primed in mixed lymphocyte cultures (MLC) in the presence of the corticosteroid methylprednisolone (MP). The generation of proliferative-, cytotoxic- and suppressor cells after specific restimulation of the cultures was studied. Low doses of MP strongly inhibited the generation of secondary proliferative and cytotoxic cells. MP did not render these cells permanently unresponsive, as lymphocytes primed in the presence of MP as were still able to respond to the original stimulators in a primary fashion. In contrast, doses of MP as high as 10 micrograms/ml which are well above clinical levels, had no inhibitory effect on the induction of suppressor cells in these cultures. Our results indicate therefore, that steroids may prevent priming of specific cytotoxic and proliferative T cells, without inhibiting the induction of suppressor lymphocytes in secondary allogeneic cultures.     

The natural history of maternal immunization against foetal platelet alloantigens

Foetomaternal alloimmune thrombocytopenia (FMAIT) occurs when maternal antibodies of an antigen-negative mother cause destruction of sensitized foetal platelets. In Caucasian populations, 6-12% of human platelet antigen (HPA)-1a-negative women develop anti-HPA-1a, and the incidence of clinically affected cases is estimated to be 10-20% of immunized women. This study was performed in order to elucidate the rate of maternal immunization, incidence of FMAIT and the likely outcome of the condition in Asians. Excluding two or more pregnancies during the period, serum samples from 24 630 pregnant women, mainly Japanese, were screened for antibodies against platelet alloantigens by means of mixed passive haemagglutination (MPHA) (Anti-HPA-MPHA, Olympus, Tokyo). Antibodies were detected in 0.91% (223/24 630) of the women's samples and the immunization rate was correlated with the number of pregnancies. Antibody specificity included anti-HPA-4b (49), anti-HPA-5a (three), anti-HPA-5b (168), anti-HPA-4b + 5b (one) and anti-Nak(a) (CD36) (two). No alloimmunization was observed within the HPA-1, HPA-2, HPA-3 or HPA-6 systems. Among HPA-4b- or HPA-5b-negative women, 24% or 14% estimated, respectively, had antibodies and 26% (10/38) or 10% (12/125) of neonates, respectively, born to these mothers developed thrombocytopenia. Two neonates born to mothers having anti-HPA-4b developed generalized purpura. No cases of intracranial bleeding or death due to FMAIT were recorded. Generalized purpura due to FMAIT occurs in one in 9359 (95% CI: 1 in 77 519-1 in 2591) pregnancies solely because of HPA-4b incompatibility.     

A first or dominant immunization. I. Suppression of simultaneous cytolytic T cell responses to unrelated alloantigens

A first or dominant immunization with one antigen markedly inhibited specific cytolytic T lymphocyte (CTL) responses to a second unrelated alloantigen without suppressing antibody responses to other antigens. Suppression was induced rapidly, became systemic, and could be transferred passively with only serum. Suppression did not result from elimination of cells capable of responding to the second antigen. The mechanisms responsible for this "priority of the first response" may be the same that help protect the fetus during pregnancy, promote renal allograft survival after multiple blood transfusions, and prevent effective CTL-mediated immunity to variants of tumor cells or infectious agents that arise during tumor progression or chronic infections.     

Factor H binds to washed human platelets.

BACKGROUND: Factor H regulates the alternative pathway of complement. The protein has three heparin-binding sites, is synthesized primarily in the liver and copurifies from platelets with thrombospondin-1. Factor H mutations at the C-terminus are associated with atypical hemolytic uremic syndrome, a condition in which platelets are consumed. Objectives The aim of this study was to investigate if factor H interacts with platelets. METHODS: Binding of factor H, recombinant C- or N-terminus constructs and a C-terminus mutant to washed (plasma and complement-free) platelets was analyzed by flow cytometry. Binding of factor H and constructs to thrombospondin-1 was measured by surface plasmon resonance. RESULTS: Factor H bound to platelets in a dose-dependent manner. The major binding site was localized to the C-terminus. The interaction was partially blocked by heparin. Inhibition with anti-GPIIb/IIIa, or with fibrinogen, suggested that the platelet GPIIb/IIIa receptor is involved in factor H binding. Factor H binds to thrombospondin-1. Addition of thrombospondin-1 increased factor H binding to platelets. Factor H mutated at the C-terminus also bound to platelets, albeit to a significantly lesser degree. CONCLUSIONS: This study reports a novel property of factor H, i.e. binding to platelets, either directly via the GPIIb/IIIa receptor or indirectly via thrombospondin-1, in the absence of complement. Binding to platelets was mostly mediated by the C-terminal region of factor H and factor H mutated at the C-terminus exhibited reduced binding.     

Transplacental immunization of the human fetus to tetanus by immunization of the mother.

Experimental studies in rats showed that immunization of the pregnant female led to the transplacental immunization of her fetuses. The possibility that this also occurred in humans was explored by immunizing 42 pregnant women with tetanus toxoid (2.5 or 5 Lf) in the fifth and eighth months of pregnancy and comparing the immune responses of their offspring with the responses of the offspring of 25 unimmunized mothers. Only the offspring of the immunized mothers were sensitized to tetanus. IgM antitetanus antibodies were in their blood before immunization with diphtheria, pertussis, tetanus vaccine (DPT), they had a more rapid (P less than 0.01) response to DPT immunization, and they were still highly sensitized (P less than 0.01) to tetanus 13 mo after birth. In addition, pregnancy had no immunosuppressive effect (P less than 0.05) on the responses of the mothers to tetanus toxoid. Thus, transplacental immunization occurs in humans; it enhances the response of the offspring to subsequent immunization, and it could be used to circumvent the necessity for immunization in early neonatal life.     

Influence of pH on stored human platelets

BACKGROUND: During storage at room temperature, platelets (PLTs) undergo several changes, a process known as PLT storage lesion. The pH is one of the variables changing and has been suggested to be a good surrogate marker for the quality of PLT concentrates. It is unknown whether the pH decrease as such induces the PLT storage lesion or that the deterioration of the PLTs results in the pH decrease. In this study, the responses of PLTs to applied pH values were investigated. STUDY DESIGN AND METHODS: PLTs were washed three times in PLT additive solution (PAS-IIIM) and were resuspended in PAS-IIIM buffers with or without 30 percent plasma with different pH values over a range from 6.0 to 7.5 (at 37 degrees C). The PLTs were stored in 50-mL culture flasks at 22 degrees C. RESULTS: During 3 days of storage in 100 percent additive solution (AS), the extracellular pH did not affect in vitro quality measures. Both at the lower and at the higher end of the pH range, we observed an increased glycolytic flux, accelerated at Day 6. Also in the presence of 30 percent plasma, the effect of extracellular pH was very limited, but all variables indicated better PLT quality with stable values up to Day 6 of storage. CONCLUSIONS: We conclude that PLTs stored in 100 percent AS are able to cope with high and low pH values without a strong deterioration within 3 days. PLTs stored in 30 percent plasma-70 percent AS are more capable in dealing with different pH values than PLTs stored in AS and remained stable for 6 days. We suggest that the pH decrease is a result of the PLT storage lesion and not the cause.     

Gene frequencies of the HPA-15 (Gov) platelet alloantigen system in Brazilians

The HPA-15 (Gov) alloantigen is a biallelic co-dominant system on human platelets, and its allele HPA-15a and HPA-15b differ by an A-->C single nucleotide polymorphism at nucleotide 2108 of the coding sequence resulting in a Tyr682Ser substitution in the mature CD109 glycoprotein. Employing the polymerase chain reaction-restriction fragment length polymorphism technique, we determined the HPA-15 gene frequencies among 276 subjects of distinct Brazilian ethnic groups including, 15 Caucasians, 15 African Brazilians, 15 Orientals, 106 Amazon Xikrin Indians, 31 Amazon Gavioes Indians and 94 blood donors. The calculated HPA-15a and HPA-15b allele frequencies found in Caucasians (0.53/0.47), African Brazilians (0.57/0.43), Orientals (0.57/0.43) and Brazilian blood donors (0.52/0.48) did not differ significantly. However, the HPA-15a and HPA-15b gene frequencies of Xikrin Indians (0.78/0.22) were significantly different from that of all other groups (P < 0.01). The HPA-15a/a, HPA-15a/b and HPA-15b/b genotype frequencies observed in Gavioes Indians were significantly different from those seen in African Brazilians (P = 0.04) and blood donors (P = 0.017). The present data showed that the distribution of the HPA-15 (Gov) system alleles observed among the Brazilian population is quite similar to the distributions already reported among Asian, Canadian and European populations. Moreover, the data indicated differences in the frequency of the HPA-15 system between Amazon Indians and other distinct Brazilian ethnic groups suggesting that Amerindians would be at higher risk of HPA-15 alloimmunization in the need of receiving blood components collected from blood donors of other ethnic groups.     

A common uptake system for serotonin and dopamine in human platelets.

Kinetic and pharmacologic properties of uptake of serotonin and dopamine by normal human platelets have been investigated to test whether platelets can be employed as a model system for the reuptake of serotonin and dopamine in brain. Uptake of serotonin into platelets closely resembles reuptake of serotonin into serotonergic neurons. In contrast, uptake of dopamine into platelets appears to be mediated inefficiently via the specific serotonin uptake mechanism, based upon several lines of evidence. Serotonin and dopamine compete with each other, Antidepressant drugs, which are competitive inhibitors of uptake of both of these neurotransmitters, act at the same concentration of drug despite large differences in the Km values. Serotonin antagonists inhibit both serotonin and dopamine uptake. Finally, a serotonin-specific uptake inhibitor (fluoxetine) blocks dopamine, as well as serotonin, uptake.     

Protein synthesis in human platelets

The platelets and leukocytes of human peripheral blood were separated and cultured for 24 h in the same medium. The culture medium was completed with S-methionine. The overall protein synthetic activity of the platelets, compared with leukocytes, was studied with a two-dimensional gel electrophoresis technique combined with fluorographic detection of labelled proteins. Our findings confirm the platelet-origin of a few newly synthesized proteins. The most pronounced newly synthesized platelet protein with a molecular weight of approximately 35 Kda and with a more acidic isoelectric point than that of actin was identified as a cytoskeleton-associated protein.     

N-acetyl-L-cysteine exerts direct anti-aggregating effect on human platelets

BACKGROUND: N-acetyl-L-cysteine, a thiol compound, has been shown to potentiate the inhibition of platelet aggregation exerted by organic nitrates and to increase the anti-aggregating effect of L-arginine, which promotes endogenous synthesis of nitric oxide (NO) acting as substrate of platelet constitutive nitric oxide synthase (NOS). It is not known whether this thiol can exert direct effects on platelet aggregability. MATERIALS AND METHODS: 14 healthy male volunteers provided platelet samples to investigate whether N-acetyl-L-cysteine directly influences platelet function and intraplatelet levels of 3',5' cyclic guanosine monophosphate (cGMP), which represents the second messenger involved in NO-induced antiaggregation. Some experiments were repeated in the presence of NOS inhibitor NG-monomethyl-L-arginine (L-NMMA), of nitric oxide-sensitive guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), of the selective cGMP phosphodiesterase inhibitor zaprinast and of calcium ionophores (A23187, ionomycin). RESULTS: N-acetyl-L-cysteine at 3000-6000 micromol L-1 decreases the responses of human platelets both in platelet-rich plasma (aggregation induced by adenosine 5-diphosphate) and in whole blood (aggregation induced by collagen). The anti-aggregating effect was prevented by preincubation with L-NMMA and guanylyl cyclase inhibitor ODQ. In resting platelets, N-acetyl-L-cysteine increased the levels of cGMP starting from a concentration of 3000 micromol L-1. Permeabilized platelets exhibited an increased sensitivity to the anti-aggregating effect of N-acetyl-L-cysteine. Also, cGMP phosphodiesterase inhibition or the increase in calcium availability, enhanced N-acetyl-L-cysteine effects on platelets. CONCLUSION: N-acetyl-L-cysteine exerts direct anti-aggregating effects through an increased bioavailability of platelet nitric oxide.     

Citrate metabolism by human platelets

As part of a study on the utilization of substrates by platelets in defined media the metabolism of citrate was measured, since citrate is a common anticoagulant of nearly all such media, and is also an intermediate of oxidative metabolism. Human platelets transferred from plasma to an artificial medium by gel filtration, were incubated with [¹⁴C]citrate at 22 °C and labelled carbon dioxide produced was measured during short-term incubations of 2 h. Citrate (1 m m ) was oxidized to carbon dioxide at low (0.3 nmol per 10⁹ platelets h⁻¹) but significant rates, and the oxidation was decreased by the presence of an alternative substrate (acetate) in the medium. There was, however, no significant conversion of citrate to glycogen. It was calculated that under normal storage conditions of platelet concentrates for transfusion purposes, the amount of citrate used cannot decrease citrate concentrations sufficiently to bring about platelet activation.     

Histamine uptake by human platelets

We have investigated the uptake of histamine by human platelets. Incubations were carried out in platelet rich plasma prepared by using sodium citrate as an anticoagulant at histamine concentrations of 2.5 nmol X 1(-1), with and without stirring, in a platelet aggregometer cuvette at 37 degrees C. Stirring increased platelet histamine uptake significantly. Conventional platelet aggregating agents (e.g. adrenaline) significantly increased platelet histamine uptake at sub-aggregatory concentrations. Histamine uptake by platelets may be a useful index of platelet behaviour when studying the effect of subaggregatory concentrations of platelet agonists in conditions where platelet aggregation is altered, e.g. peripheral vascular disease and diabetes mellitus.