Giant platelet granules in a child with the Chediak-Higashi syndrome.

Previous ultrastructural investigation have not identified abnormal lysosomes in platelets obtained from humans or animals with the Chediak-Higashi Syndrome. We report here a patient whose megakaryocytes and platelets were found to contain giant granules when viewed by light and electron microscopy. The granules measured up to 1.5 micrometer in diameter, contained either homogeneous or heterogeneous material, were acid phosphatase positive, and were present in approximately 30% of bone marrow megakaryocytes and 5% of circulating platelets. A decrease was observed in serotonin containing dense granules, serotonin uptake and serotonin release as reported previously. Microtubules in platelets and megakaryocytes were intact and no other morphologic abnormalities were identified. No clinical evidence of bleeding was observed in this patient and platelet counts have been normal. The lack of giant platelet lysosomes in other reported cases of Chediak-Higashi Syndrome attests to significant heterogeneity in this disease with a spectrum of clinical and laboratory findings.     

Medich giant platelet disorder: a unique α granule deficiency I. Structural abnormalities

Human platelet granule deficiency disorders include the gray platelet syndrome (GPS), α δ storage pool deficiency, the Hermansky–Pudlak syndrome and the White platelet syndrome. The present study describes a patient with a lifelong history of easy bleeding, thrombocytopenia and giant platelets. Her cells were found to have normal numbers of dense bodies, but a markedly decreased number of α granules. Many platelets had no α granules and resembled the gray platelets of patients with GPS. However, the empty vacuoles without granule contents that fill the cytoplasm of GPS platelets were not present in significant numbers in her platelets. In addition to the decrease in α granules the patients platelets contained membranous inclusions resembling cigars or scrolls. Usually, only one scroll open at each end was present, but many platelets contained two and some as many as five. Freeze-fracture revealed an absence of intramembranous particles in many layers of the scrolls. They occur in no other human platelet disorder, but are common in platelets from the Wistar–Furth rat. Thus, the patient is a unique variant of human platelet granule deficiency disorders unlike any described previously.     

Medich giant platelet disorder: a unique alpha granule deficiency I. Structural abnormalities

Human platelet granule deficiency disorders include the gray platelet syndrome (GPS), alpha delta storage pool deficiency, the Hermansky-Pudlak syndrome and the White platelet syndrome. The present study describes a patient with a lifelong history of easy bleeding, thrombocytopenia and giant platelets. Her cells were found to have normal numbers of dense bodies, but a markedly decreased number of alpha granules. Many platelets had no alpha granules and resembled the gray platelets of patients with GPS. However, the empty vacuoles without granule contents that fill the cytoplasm of GPS platelets were not present in significant numbers in her platelets. In addition to the decrease in alpha granules the patients platelets contained membranous inclusions resembling cigars or scrolls. Usually, only one scroll open at each end was present, but many platelets contained two and some as many as five. Freeze-fracture revealed an absence of intramembranous particles in many layers of the scrolls. They occur in no other human platelet disorder, but are common in platelets from the Wistar-Furth rat. Thus, the patient is a unique variant of human platelet granule deficiency disorders unlike any described previously.     

Thrombasthenia with an abnormal platelet membrane glycoprotein IIb of different molecular weight

We describe an individual with abnormal platelet glycoprotein (GP) IIb of different molecular weight (mol wt), a defect that distinguishes this patient from previously reported thrombasthenics. The patient, a 21-year-old female, has a mild bleeding tendency; her platelets lack adenosine diphosphate (ADP) aggregation and have severely suppressed collagen aggregation but a normal response to ristocetin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of her platelets indicates that they contain two types of GPIIb molecules: one with an abnormal mol wt (122 kd, unreduced; 128 kd, reduced) and one with a normal mol wt (128 kd, unreduced; 118 kd, reduced). Relative to the amount of GPIIb in normal platelets, her platelets contain approximately 35% abnormal GPIIb and 20% normal GPIIb. Fibrinogen binding assays on the patient's platelets indicated that they contained 25% of the normal amount of fibrinogen receptors. Crossed immunoelectrophoresis of the patient's platelets demonstrated the formation of a GPIIb/IIIa complex that was mainly composed of normal mol wt GPIIb and GPIIIa. The patient's father has decreased ADP aggregability, and his platelets also contained both abnormal and normal GPIIb (about 50% of the normal level and about 50% of the normal number of fibrinogen receptors); her mother has only normal GPIIb. These results indicate that the patient has heterozygous GPIIb molecules with an abnormality of GPIIb at the molecular level. Studies on this abnormal GPIIb should provide information about the function of GPIIb and the mechanism of its biosynthesis.     

Platelet pathology in carriers of the X-linked GATA-1 macrothrombocytopenia

Previous investigations from our laboratory have characterized the ultrastructural and cytochemical pathology of platelets in male members of a family with X-linked GATA-1 G208S macrothrombocytoenia. A large proportion of their circulating platelets were hypogranular macrothrombocytes, resembling cells from patients with the Gray Platelet Syndrome. However, most of the GATA-1 macrothrombocytes contained some alpha granules, and a small number had as many as are present in normal platelets. GATA-1 macrothrombocytes also contained tubular inclusions formed from elements of the dense tubular system wrapped around each other like scrolls. Many macrothrombocytes contained flat tubular membrane sheets connected to channels of the open canalicular system, platelets in platelets and platelets attached to platelets forming very large macrothrombocytes. The present study has examined one obligate and three potential female carriers in this family. Thin sections of their platelets examined in the electron microscope revealed features consistent with the pathology observed in male family members. Most of their platelets were normal-sized, discoid cells containing the usual complement of alpha and delta storage organelles and channels of the dense tubular system and OCS. However, a significant number of giant platelets containing the usual frequency of alpha and delta granules and hypogranular and agranular giant platelets were observed. The frequency of the macrothrombocytes varied in each of the four women studied, but were present in all. The ability of their platelets to bind multimers of vWF, in contrast to male family members, did not differ from normal controls. Near normal as well as normal platelet counts and the ability of their platelets to bind vWF multimers may protect them from the serious bleeding problems of males with the X-linked GATA-1 G208S mutation. Our findings indicate that obligate female carriers of the GATA-1 gene can be detected by examination of their platelets in the electron microscope and distinguished from the pathology of other giant platelet disorders.     

Platelet pathology in carriers of the X-linked GATA-1 macrothrombocytopenia

Previous investigations from our laboratory have characterized the ultrastructural and cytochemical pathology of platelets in male members of a family with X-linked GATA-1 G208S macrothrombocytoenia. A large proportion of their circulating platelets were hypogranular macrothrombocytes, resembling cells from patients with the Gray Platelet Syndrome. However, most of the GATA-1 macrothrombocytes contained some alpha granules, and a small number had as many as are present in normal platelets. GATA-1 macrothrombocytes also contained tubular inclusions formed from elements of the dense tubular system wrapped around each other like scrolls. Many macrothrombocytes contained flat tubular membrane sheets connected to channels of the open canalicular system, platelets in platelets and platelets attached to platelets forming very large macrothrombocytes. The present study has examined one obligate and three potential female carriers in this family. Thin sections of their platelets examined in the electron microscope revealed features consistent with the pathology observed in male family members. Most of their platelets were normal-sized, discoid cells containing the usual complement of alpha and delta storage organelles and channels of the dense tubular system and OCS. However, a significant number of giant platelets containing the usual frequency of alpha and delta granules and hypogranular and agranular giant platelets were observed. The frequency of the macrothrombocytes varied in each of the four women studied, but were present in all. The ability of their platelets to bind multimers of vWF, in contrast to male family members, did not differ from normal controls. Near normal as well as normal platelet counts and the ability of their platelets to bind vWF multimers may protect them from the serious bleeding problems of males with the X-linked GATA-1 G208S mutation. Our findings indicate that obligate female carriers of the GATA-1 gene can be detected by examination of their platelets in the electron microscope and distinguished from the pathology of other giant platelet disorders.     

Giant electron dense chains,clusters and granules in megakaryocytes and platelets with normal dense bodies: an inherited thrombocytopenic disorder IV. Ultrastructural cytochemistry and analytical electron microscopy

Platelets from a mother and son with prolonged thrombocytopenia were shown in previous studies to contain giant organelles that developed in megakaryocytes and continued to evolve in circulating cells. Whole mount platelet preparations revealed that the large organelles were electron opaque like the serotonin-rich dense bodies in normal and patient platelets, and analytical electron microscopy revealed they contained large amounts of calcium and phosphorous in a ratio close to that found in normal platelet dense bodies. However, differences in physiology, biochemistry and morphology indicated the large opaque bodies and target-organelles in patient platelets were not aberrant dense bodies. The present study has shown that the giant organelles contain peroxidase activity like primary lysosomes in polymorphonuclear (PMN) leukocytes. Further, the giant organelles in patient platelets contain acid phosphatase activity. Analytical electron microscopy demonstrated that cerium, the capture ion for the acid phosphatase reaction product, was present in the opaque organelles with calcium and phosphorous, but not present in their normal dense bodies. Since normal sized lysosomes appeared to be reduced in patient platelets, it was concluded that the large structures were abnormal lysosomes, or fused with normal platelet lysosomes during their development. Similar giant lysosomes were not present in other patient blood cells. As a result the disorder can be considered a unique lysosomal disease of platelets.     

Giant electron dense chains,clusters and granules in megakaryocytes and platelets with normal dense bodies: an inherited thrombocytopenic disorder IV. Ultrastructural cytochemistry and analytical electron microscopy

Platelets from a mother and son with prolonged thrombocytopenia were shown in previous studies to contain giant organelles that developed in megakaryocytes and continued to evolve in circulating cells. Whole mount platelet preparations revealed that the large organelles were electron opaque like the serotonin-rich dense bodies in normal and patient platelets, and analytical electron microscopy revealed they contained large amounts of calcium and phosphorous in a ratio close to that found in normal platelet dense bodies. However, differences in physiology, biochemistry and morphology indicated the large opaque bodies and target-organelles in patient platelets were not aberrant dense bodies. The present study has shown that the giant organelles contain peroxidase activity like primary lysosomes in polymorphonuclear (PMN) leukocytes. Further, the giant organelles in patient platelets contain acid phosphatase activity. Analytical electron microscopy demonstrated that cerium, the capture ion for the acid phosphatase reaction product, was present in the opaque organelles with calcium and phosphorous, but not present in their normal dense bodies. Since normal sized lysosomes appeared to be reduced in patient platelets, it was concluded that the large structures were abnormal lysosomes, or fused with normal platelet lysosomes during their development. Similar giant lysosomes were not present in other patient blood cells. As a result the disorder can be considered a unique lysosomal disease of platelets.     

Bernard-Soulier Disease: a Study of Four Patients and their Parents

S ummary. Two families with Bernard-Soulier disease, including four patients and three of their parents, were studied and detailed clinical summaries are presented. One patient in each family has suffered severe bleeding problems while the other affected sibling is less severely affected. There has been no excessive bleeding in any of the parents or other family members. The patients demonstrated the abnormalities characteristic for Bernard-Soulier disease: thrombocytopenia, giant platelets, prolonged bleeding time, abnormal platelet aggregation to human FVIII_vWF and ristocetin or bovine FVII_vWF alone, defective ristocetin-induced binding of human ¹²⁵I–FVIII_vWF multimers, decreased platelet lysis by a drug-dependent antibody and complement, and a decreased concentration of membrane glycoprotein I. The parents had normal platelet counts, bleeding times, and FVIII-mediated aggregation. However, the parents had anormally large platelets, decreased sensitivity to lysis by a drug-dependent antibody and complement, and a decreased concentration of membrane glycoprotein I. Therefore the heterozygous state for Bernard-Soulier disease is recognizable by platelet membrane abnormalities although there is no defect of platelet function and no excessive bleeding. Red cell membrane proteins of one patient were normal, suggesting that phenotypic expression of the Bernard-Soulier disease defect is restricted to platelets.     

A Cys374Tyr homozygous mutation of platelet glycoprotein IIIa (beta 3) in a Chinese patient with Glanzmann's thrombasthenia

A 20-year-old woman from a consanguineous family in the Hunan Province of the People's Republic of China was diagnosed as having Glanzmann's thrombasthenia based on (1) nearly a lifelong history of epistaxis, gum bleeding, petechiae, and purpura; (2) severe menorrhagia resulting in anemia and need for whole-blood transfusion; (3) normal coagulation assays; (4) prolonged bleeding time; (5) absent clot retraction; (6) decreased glass bead retention; (7) absent platelet aggregation in response to adenine diphosphate, epinephrine, and collagen; and (8) normal initial slope of platelet aggregation in response to ristocetin, but with a diminished maximal extent. The patient's platelets had a decreased level of platelet fibrinogen, but the deficiency was not as severe as in other Glanzmann's thrombasthenia patients. As judged by monoclonal antibody binding studies, surface glycoprotein (GP) IIb/IIIa (alpha IIb beta 3) expression was less than 15% of normal and alpha v beta 3 vitronectin receptor expression was 15% to 19% of normal, suggesting that the defect was in GPIIIa (beta 3). Immunoblotting of platelet lysates demonstrated decreased levels of GPIIb (approximately 30% to 35% of normal) and GPIIIa (approximately 10% of normal), and the GPIIb had undergone normal maturational processing into GPIIb heavy and light chains. Sequence analysis of the patient's GPIIIa RNA identified a G to A mutation at nucleotide 1219, predicting a Cys to Tyr substitution at residue 374. The patient's parents, who are first cousins, are asymptomatic and have only minor reductions in platelet aggregation. Direct sequencing of polymerase chain reaction-amplified cDNA and GPIIIa exon VIII indicated that the patient is homozygous and her parents are heterozygous for the mutation. Transient transfection studies in Chinese hamster ovary cells indicated that the mutation results in an 85% to 90% reduction in GPIIb/IIIa surface expression, but these cells retain the ability to mediate adhesion to immobilized fibrinogen. The relative preservation of platelet fibrinogen despite the very low level of platelet surface GPIIb/IIIa expression in this patient raises some interesting questions regarding the mechanism of fibrinogen uptake and the pathophysiology of Glanzmann's thrombasthenia.     

Identification of an abnormal gene for the GPIIIa subunit of the platelet fibrinogen receptor resulting in Glanzmann's thrombasthenia

The platelet fibrinogen receptor, which is composed of glycoproteins IIb (GPIIb) and IIIa (GPIIIa), belongs to a large family of receptors that participate in a multitude of biologically important adhesive interactions. Platelets from most patients with the autosomal recessive bleeding disorder, Glanzmann's thrombasthenia, are deficient in GPIIb and GPIIIa. We have used cDNA probes to analyze the GPIIb and GPIIIa genes in four patients from three kindreds with Glanzmann's thrombasthenia. Southern analysis of their DNA was identical to that observed in normals when probed with a full-length GPIIb cDNA or a 3' GPIIIa cDNA. However, in one family, a 5' 2.0 kb GPIIIa cDNA identified abnormal DNA fragments in the father and two affected siblings' genes. A series of restriction digests resulting in small genomic fragments were probed with portions of the 5' 2.0 kb GPIIIa cDNA and indicated that the abnormal sequences are flanked by normal fragments of the GPIIIa gene. To analyze further the genetic defect in this family, RNA was prepared from their platelets. Northern analysis revealed normal levels of GPIIb mRNA compared to control platelets. We were unable to identify GPIIIa mRNA of any size in the clinically affected family members. We also identified an EcoRI restriction fragment length polymorphism (RFLP) that permitted carrier status determination in the clinically unaffected siblings. These studies indicate that Glanzmann's thrombasthenia can be caused by heterogeneous defects in the GPIIIa gene. Furthermore, we have shown that platelets can be used to characterize normal and abnormal GPIIIa and GPIIb mRNA, and RFLPs may be used to determine the carrier status in some families with Glanzmann's thrombasthenia. The specific gene abnormality in this family appears to represent an example of an insertional mutation resulting in a human disease.     

Phosphotyrosine proteins in platelets from patients with storage pool disease: direct relation between granule defects and defective signal transduction

BACKGROUND AND OBJECTIVES: Storage pool diseases (SPD) are heterogeneous disorders associated with an abnormal presence of intraplatelet granules, which cause mild to moderate bleeding diathesis. We investigated signaling through tyrosine phosphorylation of proteins occurring in platelets with total or partial absence of dense- and alpha-granules in response to activation. DESIGN AND METHODS: We included a patient with severe delta-SPD, a patient with severe alpha-SPD or gray platelet syndrome, and six patients with partial deficiency of dense or a-granules. SPD was confirmed by electron microscopy evaluation of platelet ultrastructure. Platelet function was evaluated by bleeding time determination and conventional aggregometry. Platelet suspensions were activated with collagen and thrombin to analyze changes in tyrosine phosphorylation of proteins by electrophoresis and Western-blotting. RESULTS: Bleeding times were prolonged in all the patients included. Aggregation responses were slightly decreased in delta-SPD and normal in the rest of patients. Tyrosine phosphorylation in platelets from patients with partial forms of SPD was equivalent to that observed in control platelets, absent in response to collagen and thrombin activation in delta-SPD, and deficient only to thrombin activation in alpha-SPD. INTERPRETATION AND CONCLUSIONS: Tyrosine phosphorylation of proteins in activated platelets is highly dependent on the substances contained in the dense-granules and moderately dependent on those contained in the alpha-granules. A minimum amount of intraplatelet granules ensures signaling through tyrosine phosphorylation of proteins.     

Giant electron dense chains,clusters and granules in megakaryocytes and platelets with normal dense bodies: an inherited thrombocytopenic disorder III. Platelet analytical electron microscopy

A woman and her son were referred because of prolonged thrombocytopenia. Ultrastructural studies revealed the presence of giant organelles in their cells never observed previously in human platelets. Our initial reports described the evolution of two types of giant organelles in patient megakaryocytes and platelets. The last report also demonstrated that the large organelles in platelet whole mount preparations were inherently electron opaque like serotonin-rich dense bodies in normal and patient platelets. In the present study analytical electron microscopy of whole mount preparations from the patients and controls revealed that the inherently electron opaque dense and hexagonal precursor fragments, chains, clusters and giant organelles contained high concentrations of calcium and phosphorous, the major elements in normal dense bodies. The ratio of calcium to phosphorous in patient giant organelles was nearly the same as in normal platelet dense bodies. However, as shown in the previous study, levels of serotonin and adenine nucleotides were normal in patient platelets, and giant organelles and their contents were not secreted following activation of platelets with thrombin. Thus, despite the similarity in mineral content responsible for electron density, the giant organelles in patient platelets do no appear to be aberrant serotonin storage organelles.     

Immature dense granules in platelets from mice with platelet storage pool disease

Mepacrine uptake into platelets and bone marrow megakaryocytes was analyzed to further characterize the dense granule defects in a group of seven mouse pigment mutants that have characteristics of platelet storage pool disease (SPD). In contrast to our previous studies using electron microscopy, this method revealed that all mutants had normal numbers of dense granules. However, total mepacrine uptake in all mutant platelets was significantly diminished to less than 50% of normal uptake. Also, the flashing phenomenon observed when normal dense granules are irradiated with ultraviolet light was either greatly diminished or absent when platelets of individual mutants were similarly irradiated. Therefore the principal defect in the mutant platelets is an inability to accumulate dense granule contents rather than an absence of the granules. Mepacrine uptake into megakaryocytes was indistinguishable in normal and mutant mice. This indicates the mutant dense granule defects appear either very late in megakaryocyte development or early in platelet formation in correlation with development of the mature dense granule. By standard transmission electron microscopy we have not been able to detect gross structural or subcellular abnormalities in either platelets or megakaryocytes of mutant mice. It appears all seven mutants produce immature or functionally abnormal dense granules.     

A Val193Met mutation in GPIIIa results in a GPIIb/IIIa receptor with a constitutively high affinity for a small ligand

We have identified a patient designated as (GTa) with Glanzmann's Thrombasthenia (GT) diagnosed on the basis of a prolonged bleeding time and failure of the patient's platelets to aggregate. The number of glycoprotein (GP)IIb/IIIa receptors on the platelet surface was 37% of normal and those receptors displayed a defect in soluble fibrinogen binding. Nevertheless, GTa platelets showed increased adhesion to solid-phase fibrinogen and binding affinity for the RGD-mimetic (3)H-SC52012, a non-peptide GPIIb/IIIa antagonist. Dithiothreitol (DTT) and ADP enhanced the affinity for [(3)H]-SC52012 in normal platelets, but had little effect in GTa platelets. These findings suggested that GTa platelets were locked in an altered affinity state. Genetic analysis showed that GTa was a compound heterozygote for the GPIIIa gene. One allele showed a deletion at the 3' end of exon 3 resulting in a premature stop codon. The second GPIIIa allele had a G to A transition at nucleotide 577, resulting in a Val193Met substitution. HEK 293T cells transfected with mutant GPIIb/IIIaV193M bound [(3)H]-SC52012 with a higher affinity than wild-type GPIIb/IIIa, and this was not increased by DTT. The mutant receptor distinguishes between platelet adhesion and aggregation, and demonstrates the phenotype that may be expected when platelet aggregation alone is inhibited.     

Giant electron dense chains,clusters and granules in megakaryocytes and platelets with normal dense bodies: an inherited thrombocytopenic disorder III. Platelet analytical electron microscopy

A woman and her son were referred because of prolonged thrombocytopenia. Ultrastructural studies revealed the presence of giant organelles in their cells never observed previously in human platelets. Our initial reports described the evolution of two types of giant organelles in patient megakaryocytes and platelets. The last report also demonstrated that the large organelles in platelet whole mount preparations were inherently electron opaque like serotonin-rich dense bodies in normal and patient platelets. In the present study analytical electron microscopy of whole mount preparations from the patients and controls revealed that the inherently electron opaque dense and hexagonal precursor fragments, chains, clusters and giant organelles contained high concentrations of calcium and phosphorous, the major elements in normal dense bodies. The ratio of calcium to phosphorous in patient giant organelles was nearly the same as in normal platelet dense bodies. However, as shown in the previous study, levels of serotonin and adenine nucleotides were normal in patient platelets, and giant organelles and their contents were not secreted following activation of platelets with thrombin. Thus, despite the similarity in mineral content responsible for electron density, the giant organelles in patient platelets do no appear to be aberrant serotonin storage organelles.     

Increased number of pseudodrumsticks in neutrophils and large platelets. A "new' congenital leukocyte and platelet morphological abnormality

2 members of a family, a child and his father, showed a combined morphological abnormality of leukocytes and platelets. The abnormality consisted of the presence of pseudodrumsticks in the neutrophils and of large platelets. One or more than one pseudodrumstick was present in about 40% of neutrophils. Leukocyte count, differential count and enzymatic stains were normal. Large platelets constituted about 25% of the platelet population. 1 patient also had mild thrombocytopenia which appeared to be unrelated to the basic defect since it appeared after a parotitis infection. Platelet function was normal but for a moderate prolongation of the bleeding time in the patient who had mild thrombocytopenia. No chromosomal abnormality was present in the propositi. The condition seems different from other leukocyte and platelet abnormalities so far described.     

Alpha-granule pool of glycoprotein IIb-IIIa in normal and pathologic platelets and megakaryocytes

Using an immunogold staining technique and electron microscopy, we investigated the localization of the alpha-granule pool of glycoprotein (GP) IIb-IIIa in normal platelets and maturing megakaryocytes (MK), in pathologic platelets from a patient with type I Glanzmann's thrombasthenia (GT), and from three patients with the gray platelet syndrome (GPS). In normal resting platelets, GPIIb-IIIa was observed on the plasmatic side of the plasma membrane, the open canicular system (OCS) membranes, and along the internal face of the alpha-granule membrane. This location was found with three monospecific polyclonal antibodies: one anti-GPIIb-IIIa antibody, the second specific for GPIIb, and the third specific for GPIIIa. After thrombin stimulation, the alpha-granule labeling disappeared whereas membrane labeling increased. Platelets from GT did not display labeling on plasma membranes, OCS membranes, or alpha-granule membranes. Platelets from the three patients with GPS displayed intense labeling of the plasma membrane and the OCS membrane, as well as the abnormal small alpha-granules and along the inside of large vacuoles (which contain the granule membrane protein [GMP]-140). In cultured immature MK from normal progenitors, both peptide components of GPIIb-IIIa appeared in the Golgi saccules and vesicles, and in the small precursors of alpha-granules, labeling both their membranes and their matrix. It was then observed only on the membrane of the mature MK alpha-granules, although labeling was less consistent than on the platelet granules. The MK plasma membrane and demarcation membrane system also displayed GPIIb-IIIa labeling. In conclusion, this study demonstrates that GPIIb-IIIa is present on the internal face of the alpha-granule membranes of platelets (where it appears early during MK maturation) as well as in the abnormal alpha-granules of gray platelets; it is absent from GT type I platelets.     

Platelet storage pool deficiency associated with inherited abnormalities of the inner ear in the mouse pigment mutants muted and mocha

Several inherited human syndromes have combined platelet, auditory, and/or pigment abnormalities. In the mouse the pallid pigment mutant has abnormalities of the otoliths of the inner ear together with a bleeding abnormality caused by platelet storage pool deficiency (SPD). To determine if this association is common, two other mouse pigment mutants, muted and mocha, which are known to have inner ear abnormalities, were examined for hematologic abnormalities. Both mutants had prolonged bleeding times accompanied by abnormalities of dense granules as determined by whole mount electron microscopy of platelets and by labeling platelets with mepacrine. When mutant platelets were treated with collagen, there was minimal secretion of adenosine triphosphate and aggregation was reduced. Lysosomal enzyme secretion in response to thrombin treatment was partially reduced in muted platelets and markedly reduced in mocha platelets. Similar reductions in constitutive lysosomal enzyme secretion from kidney proximal tubule cells were noted in the two mutants. These studies show that several mutations that cause pigment dilution and platelet SPD are associated with abnormalities of the inner ear. Also, these mutants, like previously described mouse pigment mutants, are models for human Hermansky-Pudlak syndrome and provide additional examples of single genes that simultaneously affect melanosomes, lysosomes, and platelet dense granules.