Function of platelet mobile receptors.
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Blood Platelets↗
Biomedical subjects
J G White
Publications and source records attributed to J G White.
Detergent-resistant cytoskeleton of the surface-activated platelet differs from the suspension-activated platelet cytoskeleton.
This study contrasts the protein composition of the detergent-resistant cytoskeleton of platelets fully spread on glass with the cytoskeletal composition of resting platelets and platelets aggregated in suspension with thrombin. Complete Triton X-100-insoluble cytoskeletons were isolated from spread, resting, and suspension-activated platelets in the presence of protease inhibitors, solubilized in sodium dodecyl sulfate/EDTA and analyzed on reduced, one-dimensional polyacrylamide gels. The protein composition of the cytoskeletons differed both qualitatively and quantitatively. Most notable were more extensive incorporation of total protein, talin, and vinculin into the cytoskeleton of spread platelets than the cytoskeleton of suspension-activated platelets. Varying the concentration and time of exposure to thrombin during suspension activation did not mimic the cytoskeletal changes of surface activation. Scanning electron microscopy, measurement of lipid phosphorus content, and varying the duration of Triton extraction did not show incomplete solubilization or nonspecific trapping of constituents in the spread platelet cytoskeleton. Proteolysis of talin was minimal in suspension-activated platelets and in platelets spread for 50 minutes. The differences in the detergent-resistant cytoskeletons of surface- and suspension-activated platelets indicate significant divergence in the physiologies of platelet spreading on surfaces and platelet activation in suspension.
Adult↗
Quantification of a novel dense granule protein (granulophysin) in platelets of patients with dense granule storage pool deficiency.
An antigen-capture sandwich enzyme-linked immunosorbent assay (ELISA) was developed for a novel protein granulophysin, a constituent of the platelet dense granule (DG) membrane and used to characterize patients with dense granule storage pool deficiency (delta-SPD). The assay uses two monoclonal antibodies against the protein, one of which is conjugated to peroxidase. Purified DGs, an enriched source of the protein, were used for the standard curve. Granulophysin levels were only low in forms of delta-SPD associated with albinism. Granulophysin levels in platelet homogenates of 30 patients with the Hermansky-Pudlak syndrome form of delta-SPD were 1/4 to 1/5 of levels in controls or obligate heterozygotes. Two patients with the Chediak-Higashi form of delta-SPD syndrome also had markedly reduced levels of granulophysin. Patients with other forms of delta-SPD had normal levels of granulophysin. Two sisters with delta-SPD in one family had normal granulophysin present in empty dense granule membrane vesicles. Three members of another family with delta-SPD had low DG counts but normal granulophysin levels, indicating that in this group the level of granulophysin was maintained despite the reduction in granule formation. Thus, granulophysin quantitation facilitates characterization of delta-SPD patients and may provide clues to the nature of defective granules in delta-SPD subtypes.
Adult↗
Phencyclidine binds to blood platelets with high affinity and specifically inhibits their activation by adrenaline.
The ion channel probe phencyclidine [1-(1-phenylcyclohexyl)piperidine; PCP] selectively inhibited aggregation, secretion and ultrastructural changes in platelets induced by adrenaline, but did not affect activation induced by other common platelet agonists such as alpha-thrombin, ADP, collagen or ionophore A23187. [3H]PCP bound to platelets with high affinity (Kd 134 +/- 33 nM; 3600 +/- 1020 sites/platelet), as did the thienyl analogue [3H]TCP (1-[1-(2-thienyl)cyclohexyl]piperidine). PCP binding to platelets was increased 3-4-fold in N-methylglucamine buffer in the absence of Na+ ions. Binding was unaffected by haloperidol and was only weakly inhibited (EC50 10-20 microM), without significant stereoselectivity by the two sets of stereoselective ligands, dexoxadrol/levoxadrol and (+)MK801/(-)MK801. Binding of PCP was not competed for by adrenaline or yohimbine. Only the high-affinity binding of [3H]PCP to platelets was blocked by prior treatment of the platelets with the covalent affinity probe Metaphit, and these platelets no longer aggregated in response to adrenaline although they responded normally to alpha-thrombin, ADP and collagen. These results suggest that platelets contain high-affinity receptors for PCP that can modulate adrenaline-induced platelet activation.
Binding, Competitive↗
Mutations in the Caenorhabditis elegans unc-4 gene alter the synaptic input to ventral cord motor neurons.
Identification of the genes orchestrating neurogenesis would greatly enhance our understanding of this process. Genes have been identified that specify neuron type (for example cut and numb in Drosophila and mec-3 in Caenorhabditis elegans) and process guidance (for example, unc-5, unc-6 and unc-40 in C. elegans and the fas-1 gene of Drosophila). We sought genes defining synaptic specificity by identifying mutations that alter synaptic connectivity in the motor circuitry in the nematode C. elegans. We used electron microscopy of serial sections to reconstruct the ventral nerve-cords of uncoordinated (unc) mutants that have distinctive locomotory choreographies. Here we describe the phenotype of mutations in the unc-4 gene in which a locomotory defect is correlated with specific changes in synaptic input to a subset of the excitatory VA motor neurons, normally used in reverse locomotion. The circuitry alterations do not arise because of the inaccessibility of the appropriate synaptic partners, but are a consequence of changes in synaptic specificity. The VA motor neurons with altered synaptic inputs are all lineal sisters of VB motor neurons; the VA motor neurons without VB sisters have essentially the same synaptic inputs as in wild-type animals. The normal function of the wild-type allele of unc-4 may thus be to invoke the appropriate synaptic specificities to VA motor neurons produced in particular developmental contexts.
Animals↗
May-Hegglin anomaly: a rare cause of thrombocytopenia.
A family with four and an unrelated family with three individuals affected by the May-Hegglin anomaly are described. Platelet counts were markedly reduced and were correctly determined only in the counting chamber. Bleeding time and platelet aggregation were always normal, but platelet nucleotide concentrations (ATP and ADP) were elevated. The platelet glycoprotein complexes Ib/IX, IIb/IIIa and Ia/IIa were quantitatively normal. Platelet-associated IgG was slightly elevated, although thrombocytopenia was presumably not caused by an immunological mechanism. Morphological investigations showed giant platelets and spindle-shaped inclusion bodies in the granulocytes, while their function (phagocytic capacity, radical production) was normal. To exclude hereditary types of thrombocytopenia, morphological and family investigations are required to avoid misdiagnosis with far-reaching diagnostic and therapeutic consequences.
Blood Platelet Disorders↗
Hepatocyte function in a hollow fiber bioreactor: a potential bioartificial liver.
We have developed a novel hepatocyte loaded hollow fiber bioreactor as a potential bioartificial liver. Freshly harvested rat hepatocytes were entrapped in a three-dimensional gel matrix within hollow fibers in a perfused bioreactor. Gel entrapment allowed cells to be cultured at high density while maintaining tissue-specific function. Hepatocyte function was evaluated in 10 bioreactors, each containing approximately 5 x 10(7) cells. Oxygen consumption averaged 0.32 pmole/cell/hr, albumin appearance averaged 0.60 pg/cell/hr, and lidocaine clearance (a measure of the P-450 activity) averaged 0.74 pg/cell/hr. Function persisted for the 7 days of the study. Electron microscopy at 7 days showed the distinctive ultrastructure of viable, differentiated hepatocytes: bile canaliculi, intercellular junctions, peroxisomes, abundant mitochondria, and glycogen granules. Maintenance of tissue specific function and ultrastructure suggests that this bioreactor configuration has potential as a device to support patients in liver failure, as well as to study hepatocytes in vitro.
Amino Acids↗
Ultrastructural analysis of platelet contractile apparatus.
Methods for evaluating the organization, subcellular distribution, and rearrangements in specific constituents of the platelet contractile cytoskeleton are rapidly being developed. The procedures presented and discussed here have yielded useful information. However, a great deal more is required before the role of contractile elements of the cytoskeleton in platelet physiology will be fully understood. The new approaches employing better preservation of platelet structure and immunocytochemistry will undoubtedly provide this knowledge in the near future.
Actins↗
Influence of calcium antagonists on thrombin-induced calcium mobilization and platelet-vessel wall interactions.
Elevation of cytosolic ionized calcium plays a critical role in human platelet activation. We have evaluated three well-characterized calcium antagonists for their ability to prevent thrombin-induced calcium mobilization in Fura 2 AM-loaded platelets and also their ability to inhibit platelet-vessel wall interactions. Thrombin (0.2 U/ml) caused significant elevation of cytosolic calcium (basal 84 +/- 18, activated 546 +/- 76 nM; n = 3). Verapamil, diltiazem, and nifedipine (100 microM) did not exert any inhibitory effect on thrombin-mediated calcium elevation. Untreated platelets perfused through a Baumgartner chamber containing a rabbit aorta preparation reacted with exposed and denuded subendothelium. The percentage of the total area covered by control platelet thrombi was 39.6 +/- 3.4. Diltiazem and Nifedipine significantly reduced the percentage of area covered by platelet thrombi, but the drugs were not as effective as aspirin (8.2 +/- 1.4). Calcium antagonists studied did not inhibit thrombin-stimulated elevation of cytosolic calcium in blood platelets. Although these drugs have been shown to prevent in vitro platelet aggregation and offer some protection against risks for atherosclerosis and thrombosis, they failed to significantly inhibit platelet-vessel wall interactions leading to formation of spread platelets and aggregates.
Aspirin↗
Platelet function in adolescent idiopathic scoliosis.
Recent studies have reported abnormal platelet morphology and function in patients with adolescent idiopathic scoliosis. These abnormalities include increased platelet size and dense body numbers, abnormal aggregation, thromboxane A2 synthesis, serotonin release to adenosine diphosphate and epinephrine stimulus, and decreased myosin-adenosine-triphosphatase-specific activity. It was postulated that a membrane-specific defect in calcium transport may be partially responsible for the abnormalities found. In response to a suggestion in the literature that platelet screening could be clinically useful in scoliosis evaluation as well as in basic research of its pathophysiology, a study was performed to evaluate platelet morphology, biochemistry, and function in patients with adolescent idiopathic scoliosis. Platelets from nine volunteers with adolescent idiopathic scoliosis were compared with cells from a control group of nine patients. No significant differences in measured platelet parameters were noted between adolescent idiopathic scoliosis patients and control groups. Platelets from both groups demonstrated normal aggregation and release patterns with all agents except for a mild decreased aggregation and secretion response to epinephrine. No significant differences were noted in serotonin or adenine nucleotide levels. No significant ultrastructural differences were noted. Earlier findings of an abnormal aggregation and secretion response to adenosine diphosphate, increased numbers of dense bodies, or increased intracellular calcium could not be confirmed. On the contrary, we found normal, if not slightly decreased, numbers of dense bodies per platelet and calcium levels that were not different from controls.
Adolescent↗
Contribution of perfusion techniques to the evaluation of the hemostatic effectiveness of platelet concentrates.
Perfusion systems allowing the morphometric analysis of platelet interactions with vessel subendothelium under flow conditions have been applied to evaluate the quality and function of stored platelets. Studies performed in vitro indicate that despite the existence of storage lesions, platelets in concentrates stored for up to 5 days retain their ability to interact with the subendothelium. Perfusion studies ex vivo with nonanticoagulated blood from anemic-thrombocytopenic patients have shown the critical hemorrheological role of red blood cells facilitating platelet interactions with subendothelium. Similar studies performed on severely thrombocytopenic patients who received transfusions of platelets stored at 4 degrees C indicate that incompletely viable platelets can contribute to primary hemostasis through procoagulant mechanisms. The latter results suggest that storage lesions which contribute to impairment of platelet function may result in enhancement of platelet procoagulant activities. Perfusion techniques have contributed to the evaluation of the hemostatic effectiveness of platelet concentrates. These techniques will provide a useful model to test the impact of new storage technologies on platelet hemostatic function.
Animals↗
Ultrastructural changes in stored platelets.
A wide variety of lesions develop in blood platelets stored in vitro for long periods of time. Most of them reflect activation and damage caused by lack of gas exchange across the wall of the container and a fall in pH below 6. Newer bags facilitate O2/CO2 exchange and maintain a neutral pH. As a result, most of the lesions reflecting acidic damage or destruction have disappeared. Some storage lesions persist, however. Formation of platelets resembling doughnuts occurs in the first 24 or 48 hours after collection and concentration. They are infrequent in currently used bags. Giant alpha granules resulting from fusion of normal-sized organelles, however, are not rare. They appear as early as 4 to 5 days, even under the best conditions, and increase in frequency up to 14 days. The basis for their development remains obscure.
Animals↗
Alcohol consumption in the guinea pig is associated with reduced megakaryocyte deformability and platelet size.
Mild thrombocytopenia is common in alcoholic individuals. Ethanol appears to impair platelet production primarily by affecting the maturing megakaryocyte compartment. We recently showed that guinea pigs have an unusually large number of elongated platelet forms even under conditions of steady-state thrombopoiesis, and a portion of their mature (stage IV) megakaryocytes yield extremely long extensions on micropipette aspiration. This study evaluates the effect of a moderately low level of ethanol consumption by guinea pigs on platelet size and form and megakaryocyte deformability. Adult Duncan Hartley guinea pigs took ethanol 2.5% (vol/vol) ad libitum for 4 weeks under environmentally controlled conditions, never reaching detectable blood ethanol levels (< 0.01%); the platelet count fell 16%. Elongated platelet forms constituted 29% of the cardiac puncture platelets of control animals, but only 3% of the cardiac puncture platelets of animals given ethanol; discocytic platelets of ethanol-treated animals were also significantly smaller. Individual stage III and IV megakaryocytes were aspirated into 5 microns diameter micropipettes by stepwise increment in pressure from 10 to 200 cm water. The extensions drawn from megakaryocytes of ethanol-exposed animals were significantly shorter than the extensions from control megakaryocytes. Extremely long extensions over 50 microns in length were drawn from 21% of the control megakaryocytes but less than 1% of ethanol-exposed megakaryocytes. Moderately low level ethanol consumption was associated with reduced platelet count and size, along with rigidity of mature megakaryocytes in guinea pigs. Few ethanol-exposed megakaryocytes yielded extremely long cell extensions, nor were elongated platelet forms prevalent in the circulation of animals given ethanol.(ABSTRACT TRUNCATED AT 250 WORDS)
Alcoholism↗
Macrothrombocytopenia and progressive deafness: a new genetic syndrome.
We report a kindred with hereditary macrothrombocytopenia and progressive sensorineural hearing loss. Although the occurrence of hereditary sensorineural hearing loss associated with macrothrombocytopenia has been reported in a small number of families, varying degrees of renal pathology have always been present. In contrast to the previously reported syndromes involving a giant-platelet disorder and deafness, none of the family members in this report have had any evidence of renal dysfunction. The disorder was inherited in a linear pattern from great-grandmother to grandmother to mother to daughter. The clinical manifestations include hearing impairment that begins before the third decade and progresses to severe to profound bilateral hearing loss by the fourth decade. The platelet disorder manifests in early childhood and persists lifelong, although it tends to remain subclinical. Hematologic and ultrastructural findings will be contrasted to those found in Alport syndrome.
Blood Platelets↗
Platelet plasma membrane is equally distributed between surface and osmotically-evaginable surface-connecting membrane, independent of size, subpopulation or species.
Osmotic swelling can double the external plasma membrane surface area of normal human platelets. The surface-connected open canalicular system (SCCS) has been proposed as the major source for this additional membrane. As bovine (B) platelets have been reported to lack SCCS, we compared osmotic swelling for B and human (H) cells. Addition of water to platelet-rich-plasma (10-90% v/v) caused sequential shape change and osmotic spherocyte (OS) formation, analyzed for size and surface area changes from time-dependent phase-contrast videomicroscopic images. Selected samples were fixed and stained with tannic acid prior to osmic acid fixation for visualization of open SCCS by transmission electron microscopy. Bovine platelets required 3-4x less water dilution of PRP than human platelets, with significant OS forming at 20% water addition. Continued water dilution converted 50% of platelets to OS, with maximally stable swelling and no significant lysis for bovine OS up to 60% dilution. Electron micrographs of unactivated discocytes (D) and of optimally-swollen OS showed open SCCS in human D not detectable in any of the swollen platelets, though granules, mitochondria and a small number of vesicles and vacuoles persisted. No evidence for any open SCCS was found for bovine D or OS, though the OS otherwise appeared similar to human OS. Geometric measurements of D and nonlysed OS showed a stable, maximal 2.1 +/- 0.1 fold increase in external plasma membrane surface area with osmotic swelling, identical for different-sized normal human platelets (mean volume V = 2.8-6.8 fl), for abnormally-large platelets (V = 8.7-11.6 fl), or for bovine or rabbit platelets (V = 3-4 fl). As osmotic swelling appears to primarily externalize SCCS in human platelets, the identical relative amounts of internal membrane externalized for bovine platelets is hypothesized to arise from an osmotically more labile, "closed", and structurally simpler SCCS or from a distinct membrane source than in human platelets. It appears that the surface to invaginated ("SCCS") plasma membrane is kept constant for human, bovine and rabbit platelets, independently of platelet production, size-dependent subpopulations, or of platelet ageing.
Animals↗
Platelet activation by fMLP-stimulated polymorphonuclear leukocytes: the activity of cathepsin G is not prevented by antiproteinases.
Human polymorphonuclear leukocytes (PMN) activated by fMLP (in the presence of CaCl2, fibrinogen, and cytochalasin B) were able to induce aggregation, cytoplasmic Ca2+ increase, and thromboxane A2 production in coincubated autologousplatelets. Cell-free supernatants prepared from n-formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated PMN were able also to induce platelet activation. Antibodies against cathepsin G and different serin protease inhibitors completely suppressed the activity of PMN-derived supernatants, indicating that cathepsin G is the major platelet activator released by PMN in our system. However, antiproteinases only partially affected platelet activation induced by PMN in mixed cell suspensions. Superoxide dismutase and catalase added to the cell suspension did not affect platelet activation nor potentiated serin protease inhibitors, making a role for short-lived oxygen radicals in our experimental system unlikely. Electron microscopic observation of stirred mixed cell suspensions preincubated for 2 minutes at 37 degrees C before stimulation showed a close PMN-platelets contact without any morphologic or biochemical event suggesting platelet activation. Preincubation of the cells without stirring to minimize PMN-platelet interaction before stimulation did not modify subsequent aggregation and platelet cytoplasmic Ca2+ increase in control samples. However, in this condition trypsin inhibitor from soybean completely prevented PMN-induced platelet activation. In samples preincubated without stirring in the presence of the antiproteinase, activated PMN stuck together but platelets preserved their discoid shape and did not appear significantly activated. We propose that membrane-to-membrane contact could create a microenvironment in which cathepsin G, discharged from stimulated PMN on adherent platelets, is protected from antiproteinases.
Blood Platelets↗
Molecular analysis of an extended family with type IA (tyrosinase-negative) oculocutaneous albinism.
We have analyzed the tyrosinase coding region of three individuals having Type IA OCA within an extended family using genomic DNA amplification and dideoxy sequencing. Two of the affected individuals are dizygotic twins. All three have a common missense mutation at codon 81 (Pro----Leu) within exon I. The twins have a second missense mutation at codon 371 (Asn----Thr) within exon III and the third individual has a second missense mutation at codon 47 (Gly----Asp) within exon I. For each of these three individuals, the loss of enzyme function is the result of two different mutations, showing that they are compound heterozygotes of two mutant tyrosinase alleles.
Albinism, Oculocutaneous↗
The platelet open canalicular system: a final common pathway.
Channels of the surface-connected, open canalicular system (OCS) of human platelets serve as the pathway for transport of substances into the cells and as conduits for the discharge of alpha granule products secreted during the platelet release reaction. The purpose of the present study was to determine if both functions of the OCS can take place simultaneously. Suspensions of washed platelets were exposed to thrombin at 1 U/ml for 5, 60, or 180 seconds in the presence of fibrinogen molecules coupled to particles of colloidal gold (Fgn/Au). The samples were fixed in a low concentration of glutaraldehyde and embedded in L.R. White resin to preserve antigenicity. Thin sections were exposed to a rabbit polyclonal antibody to human fibrinogen followed by an anti-rabbit IgG coupled to 5-nm gold beads. Thrombin caused Fgn/Au particles to bind to platelets and enter channels of the surface-connected OCS. Endogenous fibrinogen detected by immunogold 5-nm beads were localized to alpha granules in resting platelets and 5 seconds after thrombin stimulation. At 60 seconds and 3 minutes Fgn/Au particles were present in swollen alpha granules, as well as OCS channels. Fibrinogen gold beads were evident in alpha granules and OCS channels connected to the platelet surface. The 18- to 20-nm Fgn/Au particles were in the same channels of the OCS as fibrinogen gold beads. The OCS is a final common pathway for uptake of particulates and discharge of secretory products in thrombin-activated human platelets.
Biological Transport↗