Alteration of release and role of adenosine diphosphate and thromboxane A2 during collagen-induced aggregation of platelets from cattle with Chediak-Higashi syndrome.

OBJECTIVE: To compare the interaction of endogenous ADP with collagen and thromboxane A(2) (TXA(2)) during collagen-induced platelet aggregation between platelets from healthy cattle and those with Chediak-Higashi syndrome (CHS). POPULATION SAMPLE: Platelets harvested from blood samples from healthy Japanese Black cattle and those with CHS. PROCEDURES: Aggregation of gel-filtered platelets; release of ATP-ADP; and generation of thromboxane B(2) (TXB(2)), a metabolite of TXA(2), were measured. RESULTS: The potency of collagen to induce aggregation in platelets of cattle with CHS (ie, CHS platelets) was less than a tenth of that in platelets of healthy cattle (ie, control platelets). Platelet aggregation induced by collagen at an intermediate concentration depended on the coexistence of ADP and TXA(2), suggesting that released ADP cannot cause platelet aggregation by itself. Collagen-induced ADP release was markedly decreased, whereas TXB(2) production was slightly low in CHS platelets, compared with that in control platelets. A combination of subthreshold amounts of ADP and 9,11-dideoxy-9alpha, 11alpha-methano-epoxy-prostaglandin F(2) (U46619), a TXA(2) analogue, caused platelet aggregation. Similarly, a combination of subthreshold amounts of collagen and ADP caused platelet aggregation, whereas collagen and U46619 were not synergistic. CONCLUSIONS AND CLINICAL RELEVANCE: Deficient ADP release ensuing from the delta-storage pool deficiency in platelets from cattle with CHS resulted in reduction of collagen-induced platelet aggregation, through attenuation of synergism between TXA(2) and ADP and between ADP and collagen. Furthermore, results of the study reported here indicated that TXA(2) was important for aggregation of bovine platelets.

Radiographic evaluation of destructive periodontal disease in blue mink in relation to age and blood morphology.

In this study, blood samples and jaws were collected from 2 genotypes of blue mink (n = 289) in order to examine phenotypic expression of specific characteristics of Chediak-Higashi Syndrome (C-HS). Blood samples were subjected to differential counts to assess the proportion of abnormal polymorphonuclear leukocytes characteristic for CH-S (C-HS-leukocytes). Abnormal leukocytes with characteristic signs of C-HS were found in blood smears from all mink included in this study. Four teeth in one half of the mandible (P3, P4, M1, M2) were subjected to quantitative radiographic evaluation of alveolar bone loss and tooth loss. There was a high prevalence of destructive periodontal disease among blue mink included in this study. Mild to moderate periodontal disease (defined by less than 50% alveolar bone loss related to 1 or more teeth) affected 73.7% of young mink (age = 7 mo) and 67.9% of older animals (age > or = 19 mo). Severe periodontal disease (defined by more than 50% bone loss related to one or more teeth) was not detected in mink aged 7 mo, but affected 15.3% of mink aged 19 mo and 39.6% of mink aged 31 mo. The positive relationship between age and periodontal disease was statistically significant (P < 0.01). The prevalence of tooth loss was found to be high among blue mink aged > 19 mo (21.6%) and was also significantly related to age (P < 0.01). A significant positive interaction between alveolar bone loss and tooth loss (P < 0.01), implies that the highly prevalent tooth loss in the mink was related to and possibly caused by destructive periodontal disease. There was no significant difference in the prevalence of periodontal disease between the 2 genotypes and age was found to be the only statistical predictor of poor production results (P < 0.01) in blue mink.

Age-related changes of the retinal pigment epithelium of cats with Chediak-Higashi syndrome.

The eyes of 7, 9, and 11-year-old Chediak-Higashi syndrome (CHS)-affected cats were examined by light, fluorescence, and electron microscopy. Numerous round to oval bodies of various sizes were associated with the retinal pigment epithelium (RPE). These bodies stained positively with periodic acid-Schiff. They also displayed a bright yellow autofluorescence and stained positively with a prolonged Ziehl-Neelsen acid-fast method for demonstration of lipofuscin, suggesting that they contained lipofuscin or a lipofuscin-like material. Ultrastructural examination disclosed the bodies to be secondary lysosomes and large to giant-sized residual bodies. Many of the residual bodies were extracellular and formed drusen-like mounds, covered by deposits of basal lamina, beneath the RPE. Also evident were scattered degenerated RPE cells and other RPE cells that had detached and migrated into the interphotoreceptor space. The presence of drusenoid bodies, and the loss of cells from the RPE monolayer in CHS eyes have not been reported previously. Many of the changes in the CHS cat eyes resemble those in non-CHS aging eyes of man and other species.

Primary and secondary lysosomes in megakaryocytes and platelets from cattle with the Chediak-Higashi syndrome.

The ultrastructure of lysosomes from megakaryocytes (MK) and platelets of cattle with the Chediak-Higashi syndrome (CHS) was characterized using acid phosphatase histochemistry with beta-glycerophosphate as substrate and cerium as a capturing agent. Acid phosphatase was localized in the trans aspect of the Golgi complex and/or granules in MK at all stages of maturation. Morphometric analysis of the diameter of each lysosome was performed on MK from CHS cattle and compared to MK from normal cattle. Lysosomes in CHS MK were neither enlarged nor different with respect to classification as secondary lysosomes, which composed 35% of the lysosomes in CHS MK. Lysosomes were demonstrated in 22% of the CHS platelet sections and appeared similar to those from normal cattle, 56% of them being classified as secondary lysosomes. Why lysosomes are not enlarged in bovine CHS MK and platelets, whereas they are enlarged in most other cell types, remains unknown.

Identification of dense granule specific membrane proteins in bovine platelets that are absent in the Chediak-Higashi syndrome.

Platelets from cattle with the Chediak-Higashi syndrome (CHS) have a platelet dense granule deficiency. One hypothesis for the platelet dense granule deficiency is that the granule is simply not formed in CHS megakaryocytes (MK). Alternative hypotheses include that the granule is assembled in CHS MK but a functional amino-nucleotide-cation storage complex cannot be formed or that the dense granule or its precursor fuses with other granules. This study was undertaken to determine if membrane proteins specific for platelet dense granules can be identified in membranes of other granules in CHS platelets. Platelets were disrupted; a mixed-granule fraction and alpha-granule enriched, mitochondrial-enriched, and dense granule-enriched subfractions were obtained. Membrane proteins in these intact granules were radiolabeled and the granule underwent hypotonic lysis. Membrane proteins were extracted from granule "ghosts", separated, and then visualized by autoradiography. Three major proteins were identified in platelet dense granule membrane subfractions. Two of these proteins could be identified in membrane extracts from the mixed-granule fraction from normal platelets. They could neither be identified in extracts from the mixed granule fraction of CHS platelets nor in membranes from alpha granule-enriched and mitochondrial-enriched subfractions. The absence of dense granule membrane proteins in membranes of other organelles within CHS platelets suggests that fusion of dense granules or its precursor with other granules cannot account for the platelet dense granule deficiency in CHS platelets.

A defect in collagen receptor-Ca2+ signaling system in platelets from cattle with Chediak-Higashi syndrome.

Decreased platelet aggregation to collagen is a cause for bleeding diathesis of Chediak-Higashi syndrome (CHS). We investigated whether the collagen receptor-Ca2+ signaling system was impaired in platelets from cattle affected with CHS. A collagen-induced increase in cytosolic Ca2+ ([Ca2+]i) was depressed in CHS platelets, which was accompanied by a decrease in the production of inositol 1,4,5-trisphosphate. When the influences of endogenous arachidonic acid metabolites and ADP were excluded, convulxin or collagen-related peptide, which are specific agonists for the collagen receptor GPVI, increased [Ca2+]i in both normal and CHS platelets. In contrast, rhodocytin, which was thought to activate another collagen receptor GPIa/IIa, increased [Ca2+]i in CHS platelets to a lesser extent than in normal ones. Cytochalasin D, an actin polymerization inhibitor, depressed the response to collagen or rhodocytin but not the response to convulxin. Adhesion of CHS platelets to acid soluble type I collagen, which was mediated by GPIa/IIa, was similar to that of normal platelets. These results suggest that a defect in the rhodocytin-sensitive pathway is responsible for decreasing the response to collagen in CHS platelets. It remains to be determined which receptor is associated with the mechanism.

Animal model. Light and electron microscopy of hepatocytes of cats with Chediak-Higashi syndrome.

Cats with a condition resembling human Chediak-Higashi syndrome (CHS) are the most recently described of five species of animals with similar syndromes. In this study, hepatocytes of cats with CHS were examined by light microscopy, histochemistry, and transmission electron microscopy. Enlarged cytoplasmic granules, morphologically consistent with lysosomes, were present in many of the CHS cat hepatocytes. The enlarged lysosomes were generally larger and more numerous in centrilobular hepatocytes and were generally larger in older cats. The lesions were similar to those reported in other species with CHS suggesting that CHS cats are a valid animal model of human CHS.

Adenine nucleotides, serotonin, and aggregation properties of platelets of blue foxes (Alopex lagopus) with the Chediak-Higashi syndrome.

Bleeding times, concentrations of serotonin in whole blood, and concentrations of adenine nucleotides as well as aggregation properties of platelets were examined in 18 blue foxes with Chediak-Higashi-like syndrome (CHS) and 16 controls. A claw of each ketamine-sedated fox was cut until bleeding started and the bleeding time was recorded as the time from the first to the last drop. The bleeding time was greatly increased in CHS foxes. Platelet counts of CHS foxes were normal, but aggregation induced by adenosine diphosphate (ADP), serotonin, collagen, and arachidonate was impaired. Adrenaline and serotonin was impaired. Adrenaline and serotonin potentiated the aggregatory effect of ADP on control as well as on CHS platelets. The mean concentration of ADP in CHS platelets was about one-third that in controls, whereas adenosine triphosphate (ATP) was approximately one-half that in controls. Serotonin could not, in most cases, be detected in blood of CHS foxes. These findings suggest that the prolonged bleeding time in the CHS foxes is, at least partly, due to a storage pool deficiency. The drastically reduced, and in some cases absent, aggregation of CHS platelets in response to arachidonate suggests that defective arachidonate metabolism contributes to the impaired hemostasis.

Restoration of neutrophil and platelet function in feline Chediak-Higashi syndrome by bone marrow transplantation.

Allogeneic bone marrow transplantation (BMT) was successfully performed in four Chediak-Higashi (CHS) syndrome affected cats. Preparatory regimens included selective intestinal flora decontamination, fractionated total body irradiation for myeloablation, and prophylactic treatment for graft-versus-host disease with cyclosporin A. Neutrophil chemotaxis under-agarose and whole-blood platelet aggregation/secretion were characterized prior to BMT and after engraftment of donor-origin marrow cells. Liver and kidney biopsies were obtained and evaluated by light and electron microscopy before, and at 6 months post-BMT to determine what effect BMT might have on abnormal lysosome fusion in hepatocytes and renal tubule cells. The platelet storage pool defect was resolved by day 40 post-BMT. In vitro neutrophil migration in all cats appeared to improve with time after BMT and complete restoration was evident by day 175 post-BMT. No apparent differences were evident in either the liver or the kidney at 6 months post-BMT. One cat developed seizures and one developed posterior paresis 5 months post-BMT; neurologic impairment ultimately resulted in death of two cats at 6 and 8 months post-BMT, respectively. Neurologic lesions in both cats were characterized by non-suppurative encephalitis. Allogeneic BMT successfully corrected the neutrophil migration defect and platelet storage pool deficiency but had no effect on lysosome distribution in liver and kidney cells of CHS cats.

Compartmentation of 4,6-difluoro-5HT studied by nuclear magnetic resonance in normal and CHS bovine platelets.

A platelet storage pool deficiency (SPD) is present in platelets from cattle with the Chediak-Higashi syndrome (CHS). The most plausible hypothesis for the SPD is that dense granule precursors are simply not formed in CHS megakaryocytes. There is, however, evidence that some recently acquired 5-hydroxytryptamine (5HT) is located in granules and that the granules have an acidic interior. To obtain a greater understanding of the processing of 5HT by SPD platelets, normal and CHS platelets were incubated with 4,6-difluoro-5HT and studied by 19F NMR at 188 mHz. Normal platelets contained 2 compartments for 4,6-difluoro-5HT as indicated by 2 well-developed resonances for each 19F. The resonances were unequal in magnitude. The predominant resonance broadened with lower temperatures and was absent in CHS bovine platelets; it was, therefore, the dense granule compartment. There was only 1 resonance for each 19F in CHS platelets. The chemical shift was identical to the minor resonance, or non-dense granule resonance, found in normal bovine platelets but the resonance width was increased, indicating that some non-dense granule 4,6-difluoro-5HT was in a more restricted environment within CHS platelets than it was in normal platelets.

Defective in vitro motility of polymorphonuclear leukocytes of homozygote and heterozygote Chediak-Higashi cats.

The in vitro migratory responses of neutrophils of homozygote and heterozygote Chediak-Higashi cats were defective in an under-agarose assay when compared to the behavior of phagocytes of control cats. The linear distances traversed by the leading front of migrating Chediak-Higashi neutrophils toward streptococcal culture supernatant, zymosan-activated serum or buffer were reduced and smaller numbers of Chediak-Higashi phagocytes populated the resulting migration areas than did cells of control animals. The relative migration parameters of the Chediak-Higashi phagocytes, however, did not differ from the corresponding parameters of control neutrophils in the presence of streptococcal culture supernatant. Therefore, phagocytes of homozygote and heterozygote Chediak-Higashi cats recognized and responded equally well to the bacterial stimuli as did cells of control animals but traveled shorter distances primarily because of a reduced inherent motility. Similar results were also obtained when the feline phagocytes were attracted by zymosan-activated serum. In addition the relative migration parameters of the neutrophils of homozygote Chediak-Higashi cats were reduced and the normalized spatial distributions of their migrating cells were significantly different in the presence of 100% and 20% zymosan-activated serum when compared to the corresponding migration parameters of carrier and control animals. Defective recognition or responses to the higher concentrations of these host-derived attractants complicated, therefore, the already reduced inherent motility of the phagocytes of homozygote Chediak-Higashi cats.

Neutropenia in cats with the Chediak-Higashi syndrome.

Thirteen cats with Chediak-Higashi syndrome and 22 control cats from the same colony, were evaluated for neutropenia. The absolute neutrophil counts of the Chediak-Higashi syndrome cats were significantly less (P less than 0.05) than those of the control cats. It is concluded that Chediak-Higashi syndrome cats, like Chediak-Higashi syndrome humans, have a neutropenia associated with the other manifestations of the syndrome. Lysozyme activity which was undetectable in the serum of both Chediak-Higashi syndrome and control cats was not of use for determining if the neutropenia was the result of neutrophil destruction.

Inheritance of Chediak-Higashi syndrome in Japanese black cattle.

Fifty-six Japanese black cattle affected with Chediak-Higashi syndrome (C-HS) have been referred to Miyazaki University Veterinary Teaching Hospital during the past 12 years, and 44 pedigree records were collected. In pedigree analysis, the parents had no clinical sign, the affected dams had clinically normal calves, and approximately equal numbers of males and females were affected, we therefore considered this syndrome to be an autosomal recessive trait. Quantitative genetic analyses were then made in a restricted area. Segregation analysis by the a priori method in 8 families showed that C-HS was a simple autosomal recessive trait. Furthermore, 36 dams and their 257 offspring (including 8 C-HS affected cattle) were analyzed using population genetics in the same area. The result was the same as in the former analysis.

Platelet dysfunction in Chediak-Higashi syndrome-affected cattle.

A serious symptom of cattle affected with Chediak-Higashi syndrome (CHS) is a bleeding tendency. This diathesis is characterized by insufficient platelet aggregation as a result of depressed response to collagen. One possible cause for the depression is a decrease in contribution of endogenous agonists such as ADP or thromboxane A(2), which are released following collagen stimulation. However, these endogenous agonists play only a minor role in collagen-induced aggregation of bovine platelets. More importantly, activation of phospholipase C as a result of a direct action of collagen is depressed, leading to a depression of Ca(2+) mobilization, in platelets from CHS-affected cattle. Several types of collagen receptor are proposed to work in concert to induce aggregation. Among them, glycoprotein VI (GPVI) and GPIa/IIa (integrin alpha2 beta1) have been supposed to play dominant roles in collagen-induced aggregation. However, there are arguments about the role of each receptor, especially the role of GPIa/IIa, and the crosstalk between receptors. Recently, we reported that the Ca(2+) signaling produced by rhodocytin, which had been first reported to be an agonist for the collagen receptor GPIa/IIa, produced much less Ca(2+) signaling in CHS platelets than in normal ones, whereas that produced by GPVI activators was normal. These suggest that GPIa/IIa or the rhodocytin-associated pathway is impaired in CHS platelets. CHS platelets are valuable to reassess the mechanism of collagen-dependent signal transduction system and to delineate the inter-relationship among collagen receptors.

Spontaneous skin lesions in beige rats (Chediak-Higashi syndrome of rats).

Beige rats, a model animal of Chediak-Higashi syndrome (CHS), frequently developed the skin lesions consisting of crust formations and alopecia in the skin around the neck from about 4 months of age. Erosion and ulceration were also observed in advance of the skin lesions. In severe cases, the lesions spread to all of the dorsum of the trunk. Skin tissues with or without lesions were studied histopathologically in 41 beige rats comparing with normal skin from 26 age-matched DA rats. Microscopically, epidermal lesions consisted of spongiosis, pustules and erosions with crust. Inflammatory cells in pustules consisted predominantly of eosinophil, and colonization of gram-positive cocci was occasionally observed in the surface area. Mites on the epidermis were also seen in some cases. Dermal lesions were superficial perivascular inflammatory cell infiltrations of eosinophils, neutrophils and mastocytes, and edema under the epidermal lesions. Follicles in the alopecic area showed resting stage and atrophic hair germ, but inflammatory changes were slight. Morphologic characters were very similar to those of chronic eosinophilic dermatitis or spongiotic dermatitis.

Inhibition of collagen-induced platelet aggregation in Japanese black cattle with inherited platelet disorder, Chediak-Higashi syndrome.

Aggregation properties of platelets were examined in Japanese Black cattle with Chediak-Higashi syndrome (CHS) and normal control cattle. Platelet aggregation induced by collagen was decreased in platelets of the cattle with CHS, but not ADP (10-20 microM), thrombin (0.5-1.0 U/ml) and phorbol-12-myristate 13-acetate (PMA, 3.2 microM). The aggregation response induced by collagen in CHS platelets lacks the change in shape which usually occurs in normal platelets. Simultaneous stimulation by collagen (10 micrograms/ml) + ADP (10 microM) is effective in restoring collagen-induced aggregating response in CHS platelet, although pretreatment of ADP (10 microM) could not restore the collagen (10 micrograms/ml)-induced aggregating response, suggesting that there is a certain threshold of stimulation intensity above which collagen-induced aggregation of CHS platelet can begin. Control normal platelets, previously exposed to ADP (10 microM) and collagen (10 micrograms/ml), showed no further response to exposure to a third aggregating agent (arachidonic acid, 5 mM). On the other hand, the final agent was able to elicit aggregating responses in CHS platelets, suggesting that the arachidonate aggregating system may be suppressed in CHS cattle, but fully activated in control animals. Furthermore, normal platelets showed a significant decrease in aggregating response to collagen when pretreated with a cyclooxygenase inhibitor, indomethacin (10(-5) M), whereas CHS platelet was insensitive to indomethacin. This indomethacin-treated normal platelet mimicked the CHS collagen-induced aggregation pattern. These data suggest that a signal transduction process from receptor-operated events to arachidonate metabolism is suppressed in collagen-induced CHS platelet aggregation.

Prolonged bleeding time in Aleutian mink associated with a cyclo-oxygenase-independent aggregation defect and nucleotide deficit in blood platelets.

A prolonged mean template bleeding time of 13 minutes was present in nine Aleutian mink affected with Chediak-Higashi syndrome (CHS) compared with 4 minutes in dark control mink. The concentrations of blood platelets in normal and affected animals did not differ significantly. However, in mink with CHS, a marked disturbance of platelet response to collagen was present. Administration of aspirin and indomethacin completely blocked CH platelet response to collagen. Blood platelet adenosine triphosphate and adenosine diphosphate values from mink with CHS were significantly less than those of normal mink, and the platelet adenosine triphosphate/adenosine diphosphate ratios were 10.31 in affected mink and 2.74 in normal mink. These findings are consistent with our previous investigations in affectd cattle and persons and indicate that a "storage pool disease" of platelets exist in the mink with CHS.

Impaired cytosolic calcium mobilization and aggregation in response to collagen in platelets from Japanese black cattle with Chediak-Higashi syndrome.

OBJECTIVE: To examine whether impaired aggregation of platelets from Japanese Black cattle with Chediak-Higashi syndrome (CHS) was attributable to mobilization of cytosolic Ca2+. ANIMALS: 4 healthy Japanese Black cattle and 3 Japanese Black cattle with CHS. PROCEDURE: Aggregation and mobilization of cytosolic Ca2+ in response to various receptor agonists was measured in platelets from healthy cattle and cattle with CHS. Involvement of endogenous ADP and arachidonic acid in collagen-induced responses was examined. Cytosolic Ca2+ concentration was measured after platelets were loaded with the Ca2+ indicator fura-PE3. Platelet aggregation was measured with an aggregometer. RESULTS: Collagen (3 to 15 micrograms/ml)-induced increases in cytosolic Ca2+ concentration and aggregation were markedly impaired for platelets from cattle with CHS, compared with values for platelets from healthy cattle. Although aggregation and the sustained phase of the cytosolic Ca2+ response to ADP were also decreased in platelets from cattle with CHS, these decreases were small, compared with those in response to collagen. A cyclooxygenase inhibitor and a phospholipase A2 inhibitor did not have any effect on peak cytosolic Ca2+ concentration or collagen-induced aggregation of platelets from healthy cattle. Responses to a P2 tau-purinoceptor antagonist suggested that decreased release of endogenous ADP was only partially involved in the impaired response to collagen among platelets from cattle with CHS. CONCLUSIONS AND CLINICAL RELEVANCE: Marked inhibition of collagen-induced Ca2+ mobilization, rather than decreased release of endogenous substances, appeared to be the major cause of impaired platelet response to collagen and the hemorrhagic tendency in cattle with CHS.

Clinical, morphologic, and biochemical characteristics of Chediak-Higashi syndrome in fifty-six Japanese black cattle.

OBJECTIVE: To characterize Chediak-Higashi syndrome (C-HS) in Japanese Black cattle. ANIMALS: 56 of 200 cattle with a bleeding disorder and giant granules in leukocytes. PROCEDURE: Clinical observation, CBC, hemostatic screening test, platelet aggregometry, electron microscopy, platelet constituent analysis, and ophthalmoscopic examination were done. RESULTS: Affected Japanese Black cattle had increased bleeding tendency and abnormal granules in their leukocytes. Susceptibility to infection was not increased. Cutaneous albinism was evident in 6 new-born calves, but not in most affected cattle. In all affected cattle, the tapetal fundus was pale and the nontapetal fundus was almost devoid of pigment. By electron microscopy, a remarkable decrease in the number of dense granules in platelets was observed. Functionally, collagen-induced platelet aggregation was markedly reduced. CONCLUSIONS: This bleeding disorder was diagnosed as C-HS. With regard to susceptibility to infection, albinism, and mortality, clinical manifestations of C-HS in Japanese Black cattle were moderate, compared with C-HS in human beings and Hereford cattle. CLINICAL RELEVANCE: Because an autosomal recessive mode of inheritance was documented and recessive homozygotes could be easily detected, C-HS in Japanese Black cattle can be controlled.

Reported causes of death of captive killer whales (Orcinus orca).

Inquiries were made to all oceanaria that maintain killer whales in North America. Causes of death determined at necropsy included mediastinal abscesses, pyometra, pneumonia, influenza, salmonellosis, nephritis, Chediak-Higashi syndrome, fungus infection, ruptured aorta, cerebral hemorrhage and a perforated post-pyloric ulcer. Captive females appear to have a higher rate of mortality than males. Growth rates for whales that died were greater than for those that survived.