Catherine Robertson

Platelet storage pool deficiency: diagnosis in patients with prolonged bleeding times and normal platelet aggregation

We evaluated 46 patients with prolonged bleeding times, and no demonstrable abnormalities of either von Willebrand factor or platelet aggregation, for possible deficiency of platelet storage pool. Studies of ATP release from thrombin‐stimulated platelets and enumeration of dense granules in platelet whole mounts were performed in these patients. Seventeen patients (35%) had both decreased ATP release and decreased numbers of dense granules, suggesting the presence of a storage pool defect. Thus, storage pool deficiency may be present in the absence of the classical aggregation abnormalities. Evidence of storage pool deficiency should be considered in all patients with an isolated unexplained prolongation of the bleeding time. The methods used in this study are readily applicable to most clinical laboratories.

Deficient Thromboxane Synthesis and Response in Platelets from Premature Infants

In vitro function of cord blood platelets from 35 premature infants (gestational age 32 ± 3.2 wk) was compared with that of 12 full-term infants and 14 adult control subjects. In comparison with adult platelets, preterm platelets showed impaired aggregation, in response to thrombin, collagen, ADP, and U46619 [a stable analog of thromboxane A2(TxA2)], and impaired [14C]serotonin secretion in response to collagen and U46619. The production of TxB2 (the stable TxA2 metabolite) in response to collagen was reduced in preterm platelets (30.2± 5.5 ng/mL) compared with full-term (52.7 ± 12.6 ng/mL) or adult control platelets (132.3 ± 38.7 ng/mL). The deficient TxB2 production and U46619 response prompted further investigation of TxA2 receptor number and binding characteristics. Immunoblotting using an anti-TxA2 receptor antibody (anti-P2) identified a single, identical 55-kD band in solubilized membranes of control, full-term, and preterm platelets. Flow cytometry using anti-P2 produced histograms that did not differ between adults and neonates. Ligand binding studies using[3H]U46619 were carried out on 10 samples from each group. Scatchard analysis yielded a single class of binding sites with no significant difference among the Kd values (85 ± 16versus 99 ± 12 versus 100 ± 12 nM) or number of binding sites per platelet (1876 ± 460 versus 2450± 478 versus 2777 ± 536) for preterm and full-term infants and adults. Therefore platelets of preterm infants show impaired TxA2 production and response. The poor response is not related to altered binding characteristics of the TxA2 receptor but may lie in the postreceptor signal transduction pathway.

The empty sack syndrome: a platelet storage pool deficiency associated with empty dense granules.

Summary. Two sisters with lifelong bleeding tendencies were examined to determine whether their condition was associated with a platelet defect. Their platelet aggregation in response to epinephrine and collagen was abnormal, and the secretion of serotonin and ATP was markedly reduced. The platelet contents of serotonin, ADP, and ATP were all diminished and the ATP:ADP ratio was increased. Direct enumeration by whole‐mount and quinacrine‐fluorescence techniques demonstrated that the platelets from both sisters had significantly fewer dense granules than controls. These characteristics are similar to an individual with Hermansky‐Pudlak syndrome and are consistent with a platelet dense granule deficiency. In contrast, immunofluorescence studies using an antibody against the dense granule membrane protein granulophysin suggested that both sisters had numbers of granules within the normal range. Evaluation by immunoblotting and ELISA indicated the presence of normal levels of granulophysin in the platelets from both sisters: FACS analysis demonstrated the surface expression of granulophysin under conditions of selective dense granule release. These results are consistent with these sisters having a form of dense granule storage pool deficiency where the granular membranes are present but the granules have reduced contents. This observation represents a novel form of storage pool disease which we have termed the empty sack syndrome.

Identification of Patients with Storage Pool Deficiency Using ATP Release and Dense Granule Counts

Recent reports have suggested that a significant number of patients with long bleeding times and normal platelet aggregation have storage pool deficiency (SPD). The present study screened 215 patients referred for evaluation of haemostasis because of a clinical bleeding history. SPD was identified by the presence of combined abnormalities in platelet ATP release and electron microscopic dense granule enumeration. Eight percent (17/215) of the patients studied were found to have SPD. Eleven percent (9/80) of patients with prolonged bleeding times had SPD, compared to 6% (8/135) of those with normal bleeding times. Two patients were identified as having both SPD and von Willebrand Disease. Although 12 of 17 SPD patients had prolonged bleeding times and/or abnormal platelet aggregation studies, five patients had neither abnormality and would not have been identified without the more specific tests for dense granule number and function.