Search

Currently, five platelet alloantigen (alloAg) systems have been established (HPA-1, -2, -3, -4, -5). Three of these are expressed on the glycoprotein (GP) IIb-IIIa complex, HPA-1, HPA-3 and HPA-4, inherited in an autosomal codominant mode. Recent investigations of the molecular basis of these platelet alloantigen systems have shown that only one nucleic acid base substitution in the genes encoding for GP IIb and GP IIIa is responsible for the polymorphism. This substitution is reflected in a difference in restriction enzyme recognition allowing platelet alloantigen typing by restriction fragment length polymorphism (RFLP) analysis of DNA amplified by the polymerase chain reaction (PCR). To validate the PCR technology for platelet typing, we have compared PCR-RFLP with monoclonal-antibody-specific immobilization of platelet antigens (MAIPA). For this purpose, we have studied different Glanzmann thrombasthenic families and particularly heterozygous individuals, who are not lacking GP IIb-IIIa, as a model to detect the occurrence of discrepancies between these two technologies. In two families, we have found differences between molecular biology and serological methods with the lack of expression of one antigen on the platelet membrane surface. In the first family, the abnormality is related to the HPA-1 alloantigen system with three informative members; in the second, the HPA-3 alloantigen system is concerned with two informative members. Considering these results, there may not always be a perfect correlation between molecular biology and serological methods, as an unknown molecular defect could interfere with the PCR results and lead to false platelet typing.(ABSTRACT TRUNCATED AT 250 WORDS)

Neonatal alloimmune thrombocytopenia (NAIT) is caused by platelet antigen incompatibility between the mother and fetus. NAIT is mainly due to alloimmunization; the frequency varying among ethnic groups. In Caucasians HPA-1a is the antigen most frequently implicated. In Japan, NAIT due to anti-HPA-4b antibody has already been described, but this is the first case to be reported in Caucasians.

We have previously demonstrated hypercoagulability utilizing a global haemostatic assay, the Overall Haemostatic Potential (OHP) in a heterogenous population of patients with a demonstrable lupus anticoagulant. Our aim in this study was to determine whether the OHP assay demonstrated a persistent hypercoagulable state in a well-defined prospective population with APLS and whether global assays were able to predict the occurrence of thrombotic complications. Informed consent was obtained and blood was collected on three occasions, three months apart from 54 patients with APLS, recruited from Haematology clinics at Royal North Shore Hospital, Sydney, Australia between May 2005 and November 2007. Clinical data was collected including history of prior and subsequent thrombotic events. Two control groups consisted of 200 healthy blood donors and 20 patients with autoimmune disorders, but no history of thrombosis. Assays performed were PT, INR, APTT, FVIIIc, lupus anticoagulant assay (LAC), anticardiolipin antibodies (ACLA), b2-glycoprotein1 antibodies (B2GP1), a thrombin generation assay (Calibrated Automated Thrombogram, CAT) and the OHP assay which utilizes thrombin (0.03 IU/ml) and rt-PA (350 ng/ml) to trigger fibrin generation and fibrinolysis, respectively, in platelet poor plasma. Change in optical density in microtitre wells is measured over 60 minutes. Statistical analysis involved calculation of means, SD, T-tests and paired T-tests utilizing SPSS v16.0. Fifty percent of APLS patients were male, compared with 10% of the autoimmune control group. APLS had been diagnosed on the basis of persistent antiphospholipid antibodies and at least one thrombotic event: VTE (n=46), ATE (n=6) or recurrent late miscarriage (n=2). Number of thrombotic events prior to study entry ranged form 1 to 6 per patient, with 49/66 events unprovoked. Samples from APLS patients on anticoagulation (OAC, n=35) were analysed separately from those not anticoagulated (n=19). Global assay results for samples collected at different time points were stable, with no significant differences on paired T-test. APLS patients had significantly shorter PT, higher fibrinogen, increased fibrin generation and reduced fibrinolysis parameters (p

No routine assays are available which quantify abnormalities in the fibrinolytic system. The Overall Haemostatic Potential (OHP) assay is a global assay of fibrin generation and fibrinolysis. Our aim in this study was to assess changes in fibrinolysis measured by the OHP assay in samples with variable levels of the different components of the fibrinolytic system: plasminogen, a2-antiplasmin, PAI-1 and TAFI. Plasmas deficient in these factors were obtained commercially. Samples were spiked with Lys-plasminogen, Glu-plasminogen, PAI-1, TAFI or pooled normal plasma to produce serial increases in these factors. Potato tuberose carboxypeptidase inhibitor (PTCI) was also used to inhibit TAFI in PNP and patient samples. OHP assays were performed utilizing either thrombin (0.03 IU/ml) or tissue factor (5 pM) to trigger fibrin generation, and rt-PA (350 ng/ml) to trigger fibrinolysis. Data analysis involved calculation of Spearman correlations for OHP assay parameters and concentrations of the various fibrinolytic factors tested. The OHP assay demonstrated markedly reduced fibrinolysis in plasminogen deficient plasmas, which normalised when plasminogen levels were increased to the normal range. At elevated Lys-plasminogen levels fibrin generation was impaired. Overall fibrinolytic potential was mildly increased in a2-antiplasmin deficiency. PAI-1 depleted plasma had mildly increased fibrinolysis compared with PNP but with addition of PAI-1, to concentrations of 300 ng/ml or higher, fibrinolysis was markedly impaired. TAFI deficient plasma had reduced fibrin generation but no change in fibrinolysis was seen in spiked samples, even with elevated TAFI levels. Representative hypofibrinolytic patient samples showed normalisation of fibrinolysis with TAFI inhibition by PTCI. The OHP assay shows appropriate changes in OFP with variations in the concentrations of activators and inhibitors of the fibrinolytic system. The assay provides a simple means of quantifying overall fibrinolysis and is sensitive to abnormalities in individual components of the fibrinolytic system. It should be useful for investigating fibrinolytic abnormalities in patients, where no routine assay is currently available.

Hypercoagulable states typically result in arterial and/or venous thromboembolism. Routine coagulation tests do not identify affected patients and thrombophilia testing detects, at best, 50% of patients at risk. We hypothesise that the OHP can differentiate patients with various hypercoagulable states from a control group. The OHP is a global haemostasis assay of fibrin generation and lysis in citrated plasma. It produces a number of parameters for comparison. We established reference ranges with plasmas from 100 normal donors, then assayed plasmas from 90 clinically hypercoagulable patients being tested for a lupus anticoagulant. A further 81 patients with demonstrable antiphospholipid antibodies (APLAs) were then assayed. Statistical analysis involved calculation of means, medians, standard deviations and Mann-Whitney testing. All parameters tested were significantly different in the clinically hypercoagulable group (p