Platelet-mediated thrombolysis in patients with δ-storage pool deficiency: a thrombelastographic analysis.

We present the first thrombelastographic descriptions of three patients with δ-storage pool deficiency, a platelet disorder that involves a deficiency of dense granules and moderate bleeding. The patients demonstrated a 49-54% loss of platelet-mediated clot strength over a 1-2-h period after normal thrombus formation. This pattern persisted, with some attenuation of loss of strength following administration of epsilon aminocaproic acid, desmopressin and platelets for tonsillectomy. Assessment of platelet function in patients with platelet granule disorders can be accomplished with thrombelastographic methods in ambulatory and perioperative settings; however, the effects of therapy for this disorder cannot be monitored with thrombelastography without obtaining a blood sample prior to prophylactic hemostatic intervention.

Down-regulation of the RUNX1-target gene NR4A3 contributes to hematopoiesis deregulation in familial platelet disorder/acute myelogenous leukemia.

RUNX1 encodes a DNA-binding α subunit of the core-binding factor, a heterodimeric transcription factor. RUNX1 is a master regulatory gene in hematopoiesis and its disruption is one of the most common aberrations in acute leukemia. Inactivating or dominant-negative mutations in the RUNX1 gene have been also identified in pedigrees of familial platelet disorders with a variable propensity to develop acute myeloid leukemia (FPD/AML). We performed analysis of hematopoiesis from 2 FPD/AML pedigrees with 2 distinct RUNX1 germline mutations, that is, the R139X in a pedigree without AML and the R174Q mutation in a pedigree with AML. Both mutations induced a marked increase in the clonogenic potential of immature CD34(+)CD38(-) progenitors, with some self-renewal capacities observed only for R174Q mutation. This increased proliferation correlated with reduction in the expression of NR4A3, a gene previously implicated in leukemia development. We demonstrated that NR4A3 was a direct target of RUNX1 and that restoration of NR4A3 expression partially reduced the clonogenic potential of patient progenitors. We propose that the down-regulation of NR4A3 in RUNX1-mutated hematopoietic progenitors leads to an increase in the pool of cells susceptible to be hit by secondary leukemic genetic events.

Role of MRP4 (ABCC4) in platelet adenine nucleotide-storage: evidence from patients with delta-storage pool deficiencies.

We previously showed that the MRP4 (ABCC4) transporter is expressed in human platelet delta-granules and may be involved in ADP transport. We now demonstrate by immunoblotting and immunofluorescence microscopy that platelet MRP4 is absent in two patients with a platelet delta-storage pool deficiency (delta-SPD)-like phenotype with reduced platelet adenine nucleotide (AN) but normal serotonin levels, whereas their other membrane marker proteins of platelet granules were normally expressed and localized. In these patients, MRP4 was present in lymphocytes, and the coding region of their MRP4/ABCC4 gene did not show any mutation that explained the lack of expression. In platelets with "classic" delta-SPD (low AN and serotonin levels), MRP4 was quantitatively (immunoblot) normal, but, like other delta-granules membrane marker proteins (eg, LAMP2), was mostly displaced from delta-granules to patches at the plasma membrane, suggesting that platelets with classic delta-SPD have an abnormality that impairs the assembly of normal delta-granules. Thus, defective expression of platelet MRP4 is associated with selective defect in AN storage. The genetic basis of the new delta-SPD phenotype remains to be elucidated.

Platelet function defects.

Inherited defects of platelet function are a heterogeneous group of disorders that can result in bleeding symptoms ranging from mild bruising to severe mucocutaneous haemorrhage. These defects may be classified according to their effect on the various steps of platelet microthrombi formation including initiation, extension and cohesion, or based on their particular structural or functional deficiency. Platelet membrane receptor deficiencies result in the rare, but well-characterized syndromes of defective clot initiation, such as Bernard-Soulier Syndrome. Platelet storage pool defects are the most common disorders affecting the extension phase of clot formation. Glanzmann thrombasthenia, with absent or dysfunctional alpha IIb beta 3 receptor is the prototypical defect of the cohesion/aggregation phase of microthrombi formation. Many of these disorders share common treatments although some therapies will have greater efficacy for one patient than another and should be individualized so as to provide optimal control of symptoms. Currently much effort is being put into methods to more rapidly and accurately diagnose patients with platelet disorders and to initiate appropriate therapy and prevent life threatening bleeding.

Platelet storage pool deficiency in Jacobsen syndrome.

Jacobsen syndrome and Paris-Trousseau Syndrome share similar congenital anomalies, thrombocytopenia, giant platelet alpha granules resulting from fusion of smaller organelles, and an 11q terminal deletion at 11q23.3. Similarities in the two cohorts have suggested that the Paris-Trousseau Syndrome is a variant of Jacobsen syndrome, or the same disorder. The present study has pointed out a significant difference between the two syndromes. Platelets from six patients with Jacobsen syndrome were markedly diminished in serotonin adenine nucleotide rich dense bodies, indicating the presence of platelet storage pool deficiency. Since platelet dense bodies are reported to be normal in size, number and distribution in the Paris-Trousseau Syndrome, the presence of platelet storage pool deficiency in six patients evaluated in the present study may distinguish the two disorders.

Hermansky-Pudlak syndrome type 3 in Ashkenazi Jews and other non-Puerto Rican patients with hypopigmentation and platelet storage-pool deficiency.

Hermansky-Pudlak syndrome (HPS), consisting of oculocutaneous albinism and a bleeding diathesis due to the absence of platelet dense granules, displays extensive locus heterogeneity. HPS1 mutations cause HPS-1 disease, and ADTB3A mutations cause HPS-2 disease, which is known to involve abnormal intracellular vesicle formation. A third HPS-causing gene, HPS3, was recently identified on the basis of homozygosity mapping of a genetic isolate of HPS in central Puerto Rico. We now describe the clinical and molecular characteristics of eight patients with HPS-3 who are of non-Puerto Rican heritage. Five are Ashkenazi Jews; three of these are homozygous for a 1303+1G-->A splice-site mutation that causes skipping of exon 5, deleting an RsaI restriction site and decreasing the amounts of mRNA found on northern blotting. The other two are heterozygous for the 1303+1G-->A mutation and for either an 1831+2T-->G or a 2621-2A-->G splicing mutation. Of 235 anonymous Ashkenazi Jewish DNA samples, one was heterozygous for the 1303+1G-->A mutation. One seven-year-old boy of German/Swiss extraction was compound heterozygous for a 2729+1G-->C mutation, causing skipping of exon 14, and resulting in a C1329T missense (R396W), with decreased mRNA production. A 15-year-old Irish/English boy was heterozygous for an 89-bp insertion between exons 16 and 17 resulting from abnormal splicing; his fibroblast HPS3 mRNA is normal in amount but is increased in size. A 12-year-old girl of Puerto Rican and Italian background has the 3,904-bp founder deletion from central Puerto Rico on one allele. All eight patients have mild symptoms of HPS; two Jewish patients had received the diagnosis of ocular, rather than oculocutaneous, albinism. These findings expand the molecular diagnosis of HPS, provide a screening method for a mutation common among Jews, and suggest that other patients with mild hypopigmentation and decreased vision should be examined for HPS.

Mild hypoxia causes severe pulmonary hypertension in fawn-hooded but not in Tester Moriyama rats.

The purpose of this study was to test whether the Tester Moriyama rat (TMR), a strain that has a serotonin platelet storage-pool deficiency similar to that of the fawn-hooded rat (FHR), develops severe pulmonary hypertension (PH) upon exposure to mild hypoxia. We compared hemodynamic parameters in catheterized 10-week-old FHR, TMR, and control Wistar rats that had been raised from birth to 10 weeks of age under normoxia (PI(O(2)) approximately 150 mmHg) or mild hypobaric hypoxia (PI(O(2)) approximately 120 mmHg). Mean pulmonary artery pressure and right ventricle to left ventricle plus septum weight ratio were much higher in the mildly hypoxic FHR compared with the normoxic FHR. These parameters were only increased slightly by exposure to mild hypoxia in the TMR and Wistar rats. Mild hypoxia did not affect mean systemic artery pressure in any of the rat strains. Exposure of FHR to mild hypoxia from 4 to 10 weeks of age did not lead to development of PH. Endothelin-1 (ET-1) mRNA and peptide levels were increased in the hypertensive lungs of mildly hypoxic FHR compared with the normotensive lungs of normoxic FHR, and of normoxic and mildly hypoxic TMR and Wistar rats. These results suggest that mild hypoxia causes severe PH and upregulation of lung ET-1 expression in neonatal FHR but not in neonatal TMR, and that the period from birth to 4 weeks of age is critical for the development of the severe PH in the FHR. A serotonin PSPD does not predispose rats to hypoxia-induced PH.

Platelet von Willebrand factor in Hermansky-Pudlak syndrome.

The Hermansky-Pudlak Syndrome (HPS) is an autosomal recessive inherited disorder characterized by oculocutaneous albinism, tissue accumulation of ceroid pigment, and a mild to moderate bleeding diathesis attributed to storage-pool deficient (SPD) platlets. Patients have platelet aggregation and release abnormalities. In addition, low levels of plasma von Willebrand factor (vWF) antigen in some HPS patients have been associated with a greater bleeding tendency than would be predicted from either condition alone. Other HPS patients have severe bleeding despite normal levels of plasma vWF, suggesting that at least one additional factor is responsible for their bleeding diathesis. Because platelet vWF levels have been well correlated with clinical bleeding times in patients with von Willebrand's disease, we have measured the platelet vWF activity and antigen levels in 30 HPS patients and have attempted to correlate their clinical bleeding with these values. The platelet vWF activity levels in patients was significantly lower than that of normal subjects (P < 0.0001). The patients as a group also had slightly lower values of plasma vWF activity when compared with normals (P-0.03). In 11 of the HPS patients, the multimeric structure of plasma vWF showed a decrease in the high molecular weight multimers and an increase in the low molecular weight multimers. In correlating the platelet and plasma vWF values with the bleeding histories, we were not able to show a predictable relationship in the majority of the patients.

TM rats: a model for platelet storage pool deficiency.

TM rats have a light brown hooded coat pattern resembling that of Fawn hooded (FH) rats which are a model of platelet storage pool deficiency (SPD). We examined whether the TM strain has the same platelet SPD as the FH strain. TM rats had a prolonged bleeding time and a low blood serotonin level, although their blood coagulation time and platelet counts were normal. The light coat color of the TM strain was judged to be associated with the red-eyed dilution gene as in the FH strain, but not pink eye dilution as in the RCS rat strain. Platelet SPD seen in TM rats may be a pleiotropic effect of the red-eyed dilution gene proposed in FH rats. Despite these similarities, the genetic background of the TM strain was obviously different from that of the FH strain. The TM strain, developed independently of the FH strain, will therefore be used as a model of platelet SPD.

Inherited prolonged bleeding time and platelet storage pool deficiency in the subtle gray (sut) mouse.

A high proportion of mouse mutants with diluted pigmentation have severely prolonged bleeding times due to platelet storage pool deficiency. The deficiency is associated with concomitant abnormalities in platelet dense granules and coat pigment granules. The coat color of the subtle gray (sut) mouse is diluted to a relatively minor degree. Analysis of platelet serotonin concentration established that this dense granule component similarly is reduced a relatively small amount in this mutant. The subtle gray mouse thus allowed a test of the hypothesis that relatively small changes in platelet dense granule contents may cause discernible increases in bleeding times. Bleeding times of mutant mice were significantly prolonged (3.4-fold) in comparison with those in normal sut/+ controls. These bleeding times were significantly reduced in comparison with other mouse pigment dilution mutants with more severe storage pool deficiency. These results establish the subtle gray mouse as an appropriate animal model for mild storage pool deficiency and human Hermansky-Pudlak syndrome. They indicate, together with related experiments, that bleeding times are highly sensitive to concentrations of platelet dense granule components such as serotonin.

Inherited platelet delta-storage pool disease in dogs causing severe bleeding: an animal model for a specific ADP deficiency.

The nature of a disorder producing moderate to severe bleeding after minor trauma, venipuncture, and surgery was studied in 3 families of American cocker spaniel dogs. In the 5 affected dogs tested, platelet counts and measurements of plasma coagulant function and von Willebrand factor were normal. However, bleeding times were prolonged in 4 of the 5 affected dogs tested, and platelet aggregation in response to ADP and collagen was consistently abnormal in 3, suggesting that the bleeding disorder was due to abnormal platelet function. Measurements of 14C-serotonin uptake and retention by the affected platelets were normal. However, their ADP content was decreased, while their ATP content was normal, resulting in a mean ATP/ADP ratio of 8.32, compared to a mean ratio of 1.9 in normal canine platelets. Electron microscopy revealed that the number and appearance of the dense granules in the affected platelets were indistinguishable from those of normal controls. These studies suggest that this bleeding disorder results from a deficient delta-granule storage pool of ADP; given the normal serotonin uptake and retention by affected platelets and the apparently normal number of dense granules, the ADP deficiency may be the consequence of a selective defect in delta-granule ADP transport. Additional studies of this unique platelet disorder will provide an opportunity to understand the mechanism of adenine nucleotide storage in platelet delta-granules.

Conditioned place preference/aversion to fenfluramine in fawn hooded and sprague-Dawley rats.

The Fawn Hooded (FH) rat strain possesses a genetic platelet storage pool deficiency which leads to an impaired capacity for platelets to store and release serotonin. While the relationship between this deficit and possible alterations in brain serotonergic levels or function remains unclear, numerous behavioral studies have indicated that FH rats exhibit differential responses to serotonergic agonists and antagonist relative to other strains. The current study used the conditioned place preference paradigm to examine the ability of fenfluramine to produce a conditioned place preference (CPP) or aversion (CPA) in FH and Sprague-Dawley (SD) rats. Results indicated that fenfluramine failed to produce CPP or CPA in SD rats, but did produce a CPA in FH rats. Results are discussed in terms of the use of conditioned place preference to assess putative differences in serotonergic functioning in FH rats.

Human platelets secrete chemotactic activity for eosinophils.

Thrombin-stimulated platelets liberate factors that induce chemotaxis of eosinophils and raise their cytosolic Ca2+ content ([Ca2+]i). The sources of this activity are the dense- and alpha-granules because inhibition of prostaglandin endoperoxide/thromboxane A2 formation and the platelet-activating factor receptor-antagonist WEB 2086 have no effect. Platelets from patients with Storage-Pool Deficiency show about 60% of the normal chemotactic activity with little effect on [Ca2+]i, whereas completely degranulated platelets fail to affect eosinophils. In concentrations secreted by the platelets, adenosine diphosphate (ADP), and platelet factor 4 have no effect, whereas adenosine triphosphate (ATP) induces a strong chemotactic response and increases [Ca2+]i. However, apart from ATP other modulating factors must be involved as platelet releasates induce more chemotaxis than ATP alone. Thus, platelets secrete factors that activate eosinophils and may contribute to inflammatory and allergic processes.

Collagen-induced rat platelet reactivity is enhanced in whole blood in both the presence and absence of dense granule secretion.

Collagen induced aggregation, ATP secretion and thromboxane (TxB2) generation of storage pool deficient platelets were compared to normal platelets of closely related rat strains. Platelet function was monitored in citrated-platelet-rich-plasma (PRP) and citrated whole blood. Wistar (W) and fawn-hooded (FH) rat strains and their F2 hybrids were utilized. The W strain, which is ancestral to the FH strain, is not storage pool deficient while the FH strain is. This was manifested by the total lack of collagen induced ATP secretion from platelets of the FH strain while the platelets of the W strain secreted normally. Utilizing platelets from the F2 generation of WxFH matings, the absence of dense granule secretion (ATP) from the FH platelets, as well as other platelet defects of FH rats, were shown to be associated with homozygosity for the red-eyed dilution gene [r]. The non-secreting FH platelets were utilized to determine the effects of secreted dense granule constituents upon collagen induced aggregation and TxB2 generation. The non-secreting storage pool deficient platelets did aggregate and did generate TxB2 upon stimulation with collagen; however, the storage pool deficient FH platelets demonstrated less TxB2 generation and did not aggregate as effectively as the normally secreting platelets of the W strain. When evaluating collagen induced platelet function in whole blood as compared to PRP, the storage pool deficient platelets remained less reactive than normally secreting platelets, but both platelet types demonstrated enhanced aggregation and increased TxB2 generation in whole blood.

Prolonged bleeding time of Chediak-Higashi cats corrected by platelet transfusion.

Cats with the Chediak-Higashi syndrome (CHS) have a platelet storage pool deficiency (SPD). Ten CHS cats were transfused with a concentrate of 51Cr-labeled platelets prepared from normal donor cats. One hour after transfusion, the donor platelet count in CHS recipient cats was 40,000-60,000/microliters. Bleeding time before transfusion was 9.1 +/- 3.0 min. When donor platelet count in CHS cats was 50,000/microliters, bleeding time was 1.7 +/- 0.2 min. Bleeding time of normal cats was 1.4 +/- 0.3 min. Bleeding time increased to 3.3 +/- 0.2 min and to 5.3 +/- 0.2 min when the platelet count was 30,000/microliters, and 15,000/microliters, respectively. The close inverse relationship between bleeding time and number of donor platelets in CHS cats (r = -0.92), suggests that prolonged bleeding time is due to a platelet abnormality, that platelet transfusion can effectively correct prolonged bleeding time in an animal model of platelet SPD and that CHS cats may be an appropriate animal model to evaluate hemostatic capabilities of transfused platelets.

Paradoxical constriction to platelets by arteries from rats with pulmonary hypertension.

We recently described the early appearance of pulmonary hypertension in the fawn-hooded rat (FHR), an animal with platelet storage pool disease also known to develop systemic hypertension at later ages. Since mediators released from aggregating platelets influence vascular tone, we hypothesized that platelet-mediated pulmonary vascular responses in FHR may be abnormal and potentially linked to the mechanism of pulmonary hypertension. To test this we examined reactivity of isolated pulmonary arteries (PA) and thoracic aortas (Ao) from young FHR with moderately severe pulmonary hypertension but normal systemic pressures. These vessels were compared with PA and Ao from control Sprague-Dawley rat (SDR). Aggregating platelets (1,000-40,000 platelets/mm3) from FHR caused dilation of SDR PA and Ao but constriction of FHR PA and Ao. Qualitatively similar responses were also observed with platelets isolated from SDR implying that abnormal responses were not simply due to the storage pool deficiency in FHR. Response to the platelet-derived endothelium-dependent vasodilator ADP was markedly impaired in FHR PA and mildly impaired in FHR Ao. Endothelium-dependent dilation to acetylcholine, but not to A23187, was mildly impaired in FHR PA while responses to both dilators were normal in FHR Ao. Endothelium-independent dilation to sodium nitroprusside was normal in both FHR PA and Ao. Constrictor sensitivity to serotonin, but not to the thromboxane A2 mimetic U-46619, was increased in FHR PA while responses to both constrictors were normal in FHR Ao. In summary, PAs from FHR with spontaneous pulmonary hypertension exhibit paradoxical constriction to both normal and storage pool deficient platelets.(ABSTRACT TRUNCATED AT 250 WORDS)

The Hermansky-Pudlak syndrome. Variable reaction to 1-desamino-8D-arginine vasopressin for correction of the bleeding time.

The effect of the synthetic vasopressin derivative 1-desamino-8D-arginine vasopressin (DDAVP = Minrin) on bleeding time was studied in nine patients with Hermansky-Pudlak syndrome; four of them were Dutch, five were Belgian. Shortening of bleeding time was observed in four of the patients with this type of storage pool disease; in one patient the response was equivocal, in two patients the response was not dramatic and in two there was no response at all. DDAVP may be useful in managing the bleeding disorder in some patients with Hermansky-Pudlak syndrome. Therefore, every patient with this syndrome should be tested with DDAVP as a preventive measure.