Light Transmission Aggregometry Does Not Correlate With the Severity of δ-Granule Platelet Storage Pool Deficiency.

Delta-granule platelet storage pool deficiency (δ-PSPD) is a poorly studied bleeding diathesis resulting from either decreased granule content or decreased average number of platelet δ-granules. Light transmission aggregometry (LTA) is commonly used to evaluate for δ-PSPD and platelet electron microscopy (EM) is used to confirm the diagnosis. Currently, little data exist examining the relationship between the likelihood of abnormal platelet aggregation findings, severity of δ-granule deficiency on platelet EM, and severity of bleeding symptoms in patients with δ-PSPD. Patients diagnosed with δ-PSPD by platelet EM who also underwent LTA testing were identified at a single institution for correlation between severity of bleeding, average number of platelet δ-granules, and number of agonist abnormalities on LTA. No statistically significant association was identified between the average number of δ-granules per platelet and likelihood of an abnormal LTA. LTA abnormalities were quite varied and only 50% diagnosed with δ-PSPD on EM had abnormal aggregation testing. Also, no correlation was seen between the number of clinical bleeding symptoms, number of average δ-granules per platelet, and the number of LTA agonist abnormalities. Our findings highlight the difficulties inherent in the laboratory evaluation of platelet function.

Molecular determinants of platelet delta storage pool deficiencies: an update.

Delta storage pool deficiency (δ-SPD) is a rare heterogeneous group of platelet disorders characterized by a reduction in the number or content of dense granules. δ-SPD causes a mild to moderate bleeding diathesis characterized mainly by mucocutaneous bleeding. Currently, no specific treatment is available and the therapeutic approach is based on prevention of excessive bleeding. However, during the last few years, important insights into the pathophysiology of δ-SPD have been achieved using mouse models and dense granule deficiency-associated congenital diseases, such as Hermansky-Pudlak syndrome and Chediak-Higashi syndrome. It thus appears that δ-SPD represents a genetically heterogeneous group of intracellular vesicle biogenesis and/or trafficking disorders. This review summarizes recent data regarding the molecular mechanisms together with clinical features of the different types of δ-SPD. Although the molecular basis of isolated inherited δ-SPD remains currently unknown, next-generation sequencing strategies should enable researchers to identify the causative genes. Identification of those genes should contribute to our understanding of the pathophysiology, represent useful tools for genetic diagnosis, and eventually lead to new specific therapeutic approaches.

An Investigation of ABO Blood Type and the Platelet Delta Granule Storage Pool.

Individuals with bleeding tendencies are more likely to have blood type O than blood types A, B, or AB. Platelet storage pool deficiencies are a lesser-known group of bleeding disorders which often go undiagnosed and may account for a significant number of patients with unexplained bleeding defects. We hypothesized that patients with platelet δ-storage pool deficiency might also have a predominance of type O blood. A retrospective review of medical records of 2,020 patients with unexplained bleeding and evaluated for δ-storage pool deficiency was performed. Correlations between dense granule numbers, blood type, and von Willebrand factor were analyzed for statistical differences. 51.5% of blood samples were blood type O compared to an incidence of 44.0% in the U.S. population. There was a significant association of vWF and blood type O but not with the delta storage pool. There is a preponderance of blood type O in the study population compared to the U.S. population. There is no statistically significant link between blood type O and lower dense granule numbers in this study.

[Congenital thrombocytopathies]. / Angeborene Thrombozytenfunktionsstörungen.

Inherited thrombocytopathies are much less frequent in comparison to acquired platelet function disorders. However, congenital disorders can lead to severe bleeding tendency and are often not diagnosed. They are induced by different platelet defects based on disorders of platelet adhesion, receptors, secretion and signal transduction. In some cases they are associated with thrombocytopenia, giant platelets and various comorbidities. This article gives an overview regarding diverse defects, their diagnosis and treatment options.

New insights into the haemostatic function of platelets.

Considerable progress has been made over the last two decades in delineating the key molecular events regulating the haemostatic function of platelets. Much of this new insight has been derived from the study of mouse models, in which the expression or structure of one or more platelet proteins has been genetically altered. Despite these advances on the research front, clinical progress in diagnosing patients with unexplained surgical bleeding or recurrent haemorrhage from mucocutaneous sites has been comparatively limited. There is a dearth of literature available to help physicians integrate and apply the burgeoning knowledge on platelet biology to diagnosing patients with atypical or unexplained platelet dysfunction. The purpose of this review is to summarise the major primary platelet disorders relevant to pathological bleeding in humans (excluding those primarily due to thrombocytopenia or acquired functional disorders), with a focus on lesions identified in mouse models that could represent candidate molecules for study in patients with impaired platelet function.

Congenital disorders associated with platelet dysfunctions.

Genetic defects of the megakaryocyte lineage give rise to bleeding syndromes of varying severity. Blood platelets are unable to fulfill their hemostatic function of preventing blood loss on vessel injury. Spontaneous bleeding is mostly mucocutaneous in nature. Most studied are deficiencies of glycoprotein (GP) mediators of adhesion (Bernard-Soulier syndrome) and aggregation (Glanzmann thrombasthenia) which concern the GPIb-IX-V complex and the integrin alphaIIbbeta3, respectively. Defects of primary receptors for stimuli include the P2Y(12)ADP receptor pathology. Agonist-specific deficiencies in the platelet aggregation response and abnormalities of signaling pathways are common and lead to trauma-related bleeding. Inherited defects of secretion from storage organelles, of ATP production, and of the generation of procoagulant activity are also encountered. In some disorders, such as the Chediak-Higashi, Hermansky-Pudlak, Wiskott-Aldrich and Scott syndromes, the molecular lesion extends to other cells. In familial thrombocytopenia (FT), platelets are produced in insufficient numbers to assure haemostasis. Some of these disorders affect platelet morphology and give rise to the so-called 'giant platelet' syndromes (MYH9-related diseases) with changes in megakaryocyte maturation within the bone marrow and premature release of platelets. Diseases of platelet production may extend to other cells and in some cases interfere with development. Transfusion of platelets remains the most common treatment of severe bleeding, management with desmopressin is common for mild disorders. Substitute therapies are available including rFVIIa and the potential use of TPO analogues for FT. Stem cell or bone marrow transplanation is being used for severe diseases while gene therapy may be on the horizon.

Defective platelet responsiveness to thrombin and protease-activated receptors agonists in a novel case of gray platelet syndrome: correlation between the platelet defect and the alpha-granule content in the patient and four relatives.

BACKGROUND: We report a novel case of gray platelet syndrome (GPS). A 14-year-old boy had bleeding diathesis, mild thrombocytopenia, giant platelets with severe defect of alpha-granule secretory proteins, myelofibrosis and splenomegaly. METHODS AND RESULTS: Platelet function studies showed a marked reduction of aggregation and Ca(2+) mobilization by thrombin, protease-activated receptor 1 (PAR1)-activating peptide (AP) and PAR4-AP, PAR1 expression at 55% of normal levels, and a more than two hundred fold reduction of in vitro whole-blood thromboxane B(2) (TXB(2)) production. Sequencing of coding regions of the PAR1 gene failed to show abnormalities. This patient was initially classified as a sporadic case of GPS, as electron microscopy failed to identify giant platelets and/or alpha-granule deficiency in his relatives. However, further studies on the father and three other relatives showed a relative lack of platelet alpha-granule proteins by immunofluorescence microscopy, a defective platelet response to PAR4-AP, and severely reduced in vitro whole-blood TXB(2) production. On this basis, we suggest that in this family, GPS was transmitted in a dominant fashion with highly variable penetrance. CONCLUSIONS: Our study suggests that current diagnostic criteria fail to identify some patients with a mild GPS phenotype and that such patients might be identified by the methods cited above. It also better characterizes the pathogenesis of defective platelet responses to thrombin, and raises interesting questions on the correlation between abnormal PAR function and the lack of alpha-granule content in GPS.

Platelet granules - secretory and secretive.

The article reviews three recent publications addressing physiological and pathological aspects of platelet granules and release as well as limitations of recent screening tests for diagnosis of non-syndromic inherited δ-storage pool disease (1-3).

Effects of mixed chimeric bone marrow repopulation on platelet storage pool-associated bleeding defects in mouse mutants.

We have previously shown mouse platelet storage pool deficiency (SPD) to be associated with lesions at eight different genetic loci, each of which is sufficient to produce murine SPD. We have also shown that normal bleeding times and normal platelet functions are restored when mice with SPD are transplanted with marrow from normal mice. Conversely, when normal mice are transplanted with mutant marrow, they present symptoms of SPD. In order to determine the amount of normal platelets needed to prevent the prolonged bleeding times associated with SPD, we established stable mixed chimeric mice by transplanting various ratios of normal and mutant marrow into lethally irradiated host animals. The proportion of normal input marrow correlated well with the proportion of normal peripheral red blood cells and platelets determined in chimerae 100 days after transplantation using direct morphology and electrophoretic variants of glucose phosphate isomerase to identify normal and mutant cell populations. The proportions of normal input marrow were also reflected in the proportions of platelets with normal and mutant platelet morphology in the chimerae. This confirms that the platelet abnormality in SPD is intrinsic to the stem cell population from which the platelets are derived. When bleeding times were determined in the mixed chimeric mice, a surprisingly high percentage of normal platelets (greater than 50% and sometimes greater than 75%) were needed to stop bleeding. These results suggest that the mutant platelets in the mixed chimeric mice may interfere with normal platelet aggregation patterns. They also raise some important considerations in devising treatment for SPD. Bleeding episodes in human SPD are normally treated by platelet transfusion. The results suggest that, at least in some cases, transfusions may not be effective. Also, in future gene therapy of this disease, it is like that a functional gene will have to be present in greater than 50% of stem cells for therapy to be effective.

Characterization of platelet function in cyclic hematopoietic dogs.

Platelet aggregation to incremental doses of eight different platelet agonists (collagen, thrombin, platelet-activating factor [PAF], arachidonic acid [AA] plus epinephrine, the calcium ionophore A23187, ADP, phospholipase C [PLC], and 12-O-tetradecanoyl phorbol-13-acetate [TPA]) was compared in normal (N) and cyclic hematopoietic (CH) dogs. Platelet aggregation was defective with collagen, PAF, TPA, and possibly thrombin as agonists but normal when ADP, PLC, arachidonic acid plus epinephrine, and A23187 were used as agonists with CH platelets. In heterozygous CH dogs, platelet aggregation was intermediately defective when tested with collagen and PAF as agonists. Thromboxane B2 (TXB2) concentrations (mean +/- SD; pg/10(6) platelets), as measured by RIA, were similar in CH and normal dogs both prior to (CH: 7.6 +/- 7.0; N: 5.5 +/- 3.9) and after collagen stimulation (collagen: 141.3 +/- 42.5; 123.1 +/- 38.4). Granule storage pools of serotonin and platelet adenine nucleotides were markedly decreased in homozygous CH but not heterozygous CH dogs. Thrombin stimulated phosphorylation of 40- and 20-kd proteins in platelets from CH and normal dogs to an equal extent. However, collagen-stimulated phosphorylation of the 40- but not the 20-kd protein was significantly decreased in platelets from CH dogs. These data suggest that there is a biochemical defect in platelets from CH dogs that results in storage pool disease and decreased phosphorylation of a 40-kd protein.

Use of a microchip flow-chamber system as a screening test for platelet storage pool disease.

Platelet storage pool disease (SPD) is a platelet function disorder characterized by a reduction in the number or content of α-granules, dense granules, or both, and is diagnosed by specialized tests. Patients with SPD often present with prolonged bleeding time (BT), but the sensitivity and reproducibility of this test have limitations, often resulting in false negatives. It has recently been reported that an automated microchip flow-chamber system (T-TAS(®)) is useful in the assessment of anti-platelet therapy, and could have potential as a screening test for SPD. We examined the utility of T-TAS in three individuals from one family diagnosed with δ-SPD. The propositus had a mildly prolonged BT, and the standard tests for platelet function were close to the normal range. Whole blood samples were anti-coagulated with hirudin and applied to T-TAS microchips coated with collagen (PL-chips) at shear rates of 1000 and 2000 s(-1). Platelet thrombus formation (PTF) was monitored with a pressure sensor. Markedly depressed PTF was observed in all cases at both shear rates. These findings indicate that T-TAS is highly sensitive to the defect in these patients with SPD, and may represent a good candidate screening test for a wide range of platelet function disorders.

Electron-dense chains and clusters in platelets from patients with storage pool-deficiency disorders.

Human platelets are known to contain inherently electron-opaque dense bodies, the storage sites for adenine nucleotides and serotonin, which can be identified under the electron microscope without fixation or staining. Recently we have reported that platelets also contain chains and clusters that are also opaque when examined by the whole-mount technique. The possibility that the newly described structures might be stages in the development of dense bodies requires consideration. The present study examined platelets from patients with Hermansky-Pudlak syndrome, Chediak-Higashi syndrome and platelet storage pool disease, whose cells are markedly deficient or lack dense bodies. Platelets from these individuals contained the same frequency of chains and clusters as found in platelets from normal controls. Thus, chains and clusters do not appear related to formation of dense bodies.

Assessment of lumiaggregometry for research and clinical laboratories.

Platelet aggregometry is often used to help diagnose storage pool disease (SPD-reduced amounts of granule nucleotides) and release defects (abnormal release of granule nucleotides). The general assumption that normal aggregation patterns are sufficient to rule out the diagnosis of one of these disorders has been invalidated by the recent publication of two papers describing patients with clinical bleeding, prolonged bleeding times and normal aggregation patterns in spite of defective release. The lumiaggregometer provides a tool for measuring platelet release and aggregation simultaneously. This paper presents a standardized, reproducible method for the use of the lumiaggregometer based on a "standard curve". Data obtained during the development of the procedure are presented including normal ranges of release at different concentrations of agonists, release measured in intrinsic disorders as well as in patients on aspirin, and values for release relative to varying platelet counts. A monoclonal antibody (anti-p24/CD9; MAb7) which activates platelets similarly to thrombin and may be a useful reagent for distinguishing SPD and release defects is also introduced.

Secreted dense granule adenine nucleotides promote calcium influx and the maintenance of elevated cytosolic calcium levels in stimulated human platelets.

Evidence that secreted dense granule adenine nucleotides mediate part of the agonist-induced cytosolic calcium ([Ca2+]i) responses in human platelets was obtained from comparisons of fura-2-loaded platelets from normal subjects and from patients with a form of platelet storage pool deficiency (SPD) in which the secretory dense granules and their contents are virtually absent. SPD platelets had normal initial [Ca2+]i increases induced by thrombin and the endoperoxide analog U46619, but a significantly enhanced decay of elevated [Ca2+]i levels following the initial increases. With thrombin, this enhanced [Ca2+]i decay was associated with decreased Ca2+ influx, as measured by Mn2+ quench of fura-2 fluorescence. Addition of micromolar concentrations of ADP, alone or together with ATP, after stimulation reversed the enhanced [Ca2+]i decay and increased Mn2+ quench in SPD platelets, but had no effect on these responses in normal platelets, while addition of 100-fold higher concentrations of ATP or apyrase before stimulation increased [Ca2+]i decay and decreased Mn2+ quench in normal platelets, but had little effect in SPD platelets. ATP and alpha,beta-methylene ATP, a specific agonist for P2X1 receptors, at micromolar concentrations also increased Mn2+ quench, but to lesser extents than did ADP, in SPD platelets isolated and loaded with fura-2 in the presence of apyrase. Similar effects of ADP and excess ATP were seen in U46619-stimulated platelets, but decreased Ca2+ influx could not be measured directly in SPD platelets, presumably due to the very transient influx response seen with U46619. These results suggest that secreted dense granule ADP and ATP contribute to the maintenance of elevated [Ca2+]i levels, but not to the initial [Ca2+]i increases, in stimulated human platelets, most likely via a nucleotide-specific component of Ca2+ influx which may be mediated by interactions with both P2X1 and P2Y1 purinoceptors.

Human platelets deficient in dense granules contain normal amounts of pp60c-src.

We have determined that pp60c-src, a protein tyrosine kinase abundant in normal platelets, is present at comparable levels in platelets that are deficient in dense granules (Hermansky-Pudlak syndrome). Relative quantitation of pp60c-src was performed by immunoblot analysis after protein separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Our data suggest that human platelet dense granules, unlike chromaffin cell secretory granules, are not the major intracellular site of localization of pp60c-src.

Platelet membrane defects in fawn hooded bleeder rats.

An inbred strain of fawn hooded rats with a congenital platelet defect shows a marked bleeding tendency with prolonged bleeding time. This haemorrhagic disorder has been exclusively related to a deficiency of nucleotides in platelet dense granules. When tested in cell electrophoresis platelets from fawn hooded bleeder rats showed a significantly lower electrophoretic mobility than normal rat platelets. Subsequent studies on the platelet membrane protein pattern by high resolution two-dimensional gel electrophoresis revealed the deficiency of a membrane glycoprotein (apparent molecular mass 90.000, isoelectric point 5.6), which is detectable in normal rat platelets after surface labeling by periodate-tritiated sodium borohydride. It seems likely, that this glycoprotein defect contributes at least partially to the disorder of platelet function in fawn hooded bleeder rats.

Diagnosis of storage pool deficiency by determination of diadenosine 5', 5'''-P1,P4-tetraphosphate in whole blood.

Platelets from cat and cattle with Chediak-Higashi disease were found completely devoid of Ap4A as measured by high performance liquid chromatography. Using a very sensitive firefly biolumnescence method 6% of the normal content of Ap4A was, however, found in platelets from sick animals. A content of Ap A of 1.90 +/- 0.11 X 10 M (means +/- SEM, n = 10) was found in whole normal human blood as measured by firefly bioluminescense method in trichloroacetic acid extracts of the blood samples. This concentration corresponds to the contribution from the platelets, thus the contribution of Ap4A from erythrocytes and the "buffy-coat" is negligible. Using the same method an Ap4A contents in platelets of 0.063 and 0.021 nmol/mg of protein compared to the normal content of 0.42 nmol/mg of protein (1) was found in two patients with severe myeloproliferative disorder calculated in this way on basis of platelet counts and on the assumption that 10(11) platelets contain 189 mg of protein (2). Comparison of these figures with parallel HPLC analyses on acid extracts of platelets isolated from the same patient were in agreement. The storage pool deficiency of adenine nucleotides in this disease found by others on basis of release experiments (3) can thus be diagnosed by rapid and simple measurements of Ap4A in whole blood using the advantage of Ap4A being a specific components stored in dense granules.