Physical, chemical and functional changes following platelet activation in normal and "giant" platelets

Unactivated discocytes in healthy human donors have mean volumes of approximately 6.0 microns3 (range 3.8-7.5 microns3), while mean values for similarly-shaped discocytes obtained from donors with the hereditary "giant" platelet syndromes were either normal (one Bernard-Soulier syndrome (BSS) and all five members of a family with the Montreal platelet syndrome (MPS) or, on average, up to twice normal (range 6.4-13.8 microns3). This apparent heterogeneity is complicated by the much more consistent and significant observation that both BSS and MPS platelets undergo a defective hypervolumetric shape change following activation which is prolonged indefinitely, in contrast to a transient hypervolumetric change measureable in 1-5 s following ADP addition to normal platelets. It is suggested that the hypervolumetric shape change in both normal and "giant" platelets is accompanied by an increase in externalized plasma membrane surface area, with the most probable source being surface-connected canalicular system. Membrane glycoprotein I abnormalities were not detectable in platelets for 2/3 sibling MPS donors. The precise relation of these membrane changes to altered platelet functions is compared for normal and "giant" platelets, but largely remains to be experimentally determined. Early shape change appears tightly associated with early microscopically-measured aggregation (PA), with both PA and turbidimetrically-measured macroaggregation generally appearing normal to elevated for "giant" platelets.     

Redistribution of alpha-granules and their contents in thrombin- stimulated platelets

The redistribution of beta-thromboglobulin (beta TG), platelet Factor 4 (PF4), and fibrinogen from the alpha granules of the platelet after stimulation with thrombin was studied by morphologic and immunocytochemical techniques. The use of tannic acid stain and quick-freeze techniques revealed several thrombin-induced morphologic changes. First, the normally discoid platelet became rounder in form, with filopodia, and the granules clustered in its center. The granules then fused with one another and with elements of the surface-connected canalicular system (SCCS) to form large vacuoles in the center of the cell and near the periphery. Neither these vacuoles nor the alpha granules appeared to fuse with the plasma membrane, but the vacuoles were connected to the extracellular space by wide necks, presumably formed by enlargement of the narrow necks connecting the SCCS to the surface of the unstimulated cell. The presence of fibrinogen, beta TG, and PF4 in corresponding large intracellular vacuoles and along the platelet plasma membrane after thrombin stimulation was demonstrated by immunocytochemical techniques in saponin-permeabilized and nonpermeabilized platelets. Immunocytochemical labeling of the three proteins on frozen thin sections of thrombin-stimulated platelets confirmed these findings and showed that all three proteins reached the plasma membrane by the same pathway. We conclude that thrombin stimulation of platelets causes at least some of the fibrinogen, beta TG, and PF4 stored in their alpha granules to be redistributed to their plasma membranes by way of surface-connected vacuoles formed by fusion of the alpha granules with elements of the SCCS.     

Morphologic and hematologic characteristics of storage pool deficiency in beige rats (Chédiak-Higashi syndrome of rats)

Characterization of beige rats as having a platelet storage pool deficiency (SPD) was undertaken. Platelets from beige rats, an animal model of Chédiak-Higashi syndrome (CHS), completely lacked the ability to aggregate when stimulated with high dosages of collagen (50 micrograms/ml), and lacked secondary aggregation induced by adenosine diphosphate (ADP). Concentrations of ADP, ATP, and serotonin in the platelets of beige rats were significantly lower than those of control rats. However, platelet count remained within normal values. Electron microscopy revealed that platelets had fewer dense granules, whereas other organelles had normal structure. This morphologic and functional evidence confirms that platelets of beige rats have the typical characteristics of SPD.     

PLATELET PHAGOCYTOSIS AND AGGREGATION

The addition of latex particles to native (no anticoagulant) or citrated human platelet-rich plasma (PRP), or to a once-washed platelet suspension causes platelet aggregation. This aggregation is associated with phagocytosis of the latex particles by the platelets and appears to be due to release of adenosine diphosphate (ADP) from the platelets. Adenosine and adenosine monophosphate, which are known to inhibit platelet aggregation induced by ADP, also block that induced by latex. These compounds do not prevent the phagocytosis of latex particles by the platelet. The addition of iodoacetate and 2,4-dinitrophenol in appropriate concentrations to the PRP, prior to the addition of the latex, blocks platelet aggregation and phagocytosis. This is also true for the chelating agent ethylenediaminetetraacetate (EDTA). Platelets left in contact with latex for a sufficient period of time show loss of their granules. Leucocytes phagocytose both latex and platelets that had themselves phagocytosed latex. It is concluded that phagocytosis of latex particles by platelets resembles that by white cells, and that in both processes metabolic changes appear to be involved.     

Stimulatory effects of endotoxin on the platelet secretory process

The effects of the lipopolysaccharides (LPS) of Proteus mirabilis (smooth and rough types), differing significantly in their composition on the release of compounds stored in specific platelet granules, were studied. There are two main types of secretory granules in blood platelets. Dense granules contain adenine nucleotides, and in alpha-granules different proteins are stored. The LPS were found to cause a dose-dependent release of proteins and adenine nucleotides. In the extracellular medium LDH activity was not present. The results presented in this study indicate that LPS from P. mirabilis act directly on blood platelets and induce the platelet secretory process. In comparison with thrombin, a strong platelet agonist, the action of the endotoxins tested was weak.     

Impaired platelet activation in familial high density lipoprotein deficiency (Tangier disease)

ATP binding cassette transporter A1 (ABCA1) is involved in regulation of intracellular lipid trafficking and export of cholesterol from cells to high density lipoproteins. ABCA1 defects cause Tangier disease, a disorder characterized by absence of high density lipoprotein and thrombocytopenia. In the present study we have demonstrated that ABCA1 is expressed in human platelets and that fibrinogen binding and CD62 surface expression in response to collagen and low concentrations of thrombin, but not to ADP, are defective in platelets from Tangier patients and ABCA1-deficient animals. The expression of platelet membrane receptors such as GPVI, alpha2beta1 integrin, and GPIIb/IIIa, the collagen-induced changes in phosphatidylserine and cholesterol distribution, and the collagen-induced signal transduction examined by phosphorylation of LAT and p72syk and by intracellular Ca2+ mobilization were unaltered in Tangier platelets. The electron microscopy of Tangier platelets revealed reduced numbers of dense bodies and the presence of giant granules typically encountered in platelets from Chediak-Higashi syndrome. Further studies demonstrated impaired release of dense body content in platelets from Tangier patients and ABCA1-deficient animals. In addition, Tangier platelets were characterized by defective surface exposure of dense body and lysosomal markers (CD63, LAMP-1, LAMP-2, CD68) during collagen- and thrombin-induced stimulation and by abnormally high lysosomal pH. We conclude that intact ABCA1 function is necessary for proper maturation of dense bodies in platelets. The impaired release of the content of dense bodies may explain the defective activation of Tangier platelets by collagen and low concentrations of thrombin, but not by ADP.     

Ultrastructural morphometric observations on serial sectioned human blood platelet subpopulations

The relationships between the volume of human platelets and their cytoplasmic organelles were studied by morphometric analysis. Platelets were separated into four density-dependent subpopulations on an arabino-galactan gradient. In vitro activation of platelets was effectively prevented by maintaining them at a constant ambient temperature of 37 degrees C. Serial sections were cut through platelets, morphometrically analyzed and the platelets reconstructed. The volumes of the individual platelets and their constituent granules, mitochondria and open canalicular systems (OCS) were calculated. Individual organelles were counted. The mean volumes of the platelets of the subpopulations decreased significantly as density decreased (p = 0.01). Also, as the density of platelets decreased, there was a decrease in the mean unit volume of their granules (p = 0.003). In contrast, independent of platelet volume or density, the OCS occupies about 10% of the platelet volume. These findings indicate that it is possible to prevent in vitro platelet activation by maintaining their environment at 37 degrees C. Our study confirms the direct relationships between platelet volume and density; and platelet density and granule content. There is no ready explanation for the constant relationship between platelet volume and that of the OCS.     

Characterization of platelet abnormalities of Tester Moriyama (TM) rats with storage pool deficiency

Platelet abnormalities of Tester Moriyama (TM) rats, which have prolonged bleeding time with normal platelet count, were characterized by comparison with those of fawn-hooded (FH) rats with platelet storage pool deficiency (SPD). Morphologically, the dense granules were virtually lacking in platelets from TM and FH rats. Platelets from TM and FH rats aggregated in response to adenosine diphosphate (ADP), but failed to have secondary aggregation. In contrast, platelet aggregation was completely absent in response to 1 to 20 micrograms of collagen/ml, although partial aggregation was observed at the higher dosage of 50 micrograms/ml. Normal amounts of platelet membrane glycoproteins IIb/IIIa were expressed in TM and FH rats, but platelet adenosine triphosphate (ATP) and ADP contents were lower than those in platelets from control Wistar rats. Platelet ATP-to-ADP ratio of TM and FH rats was significantly higher than that of Wistar rats. Serotonin content in platelets from TM and FH rats was 20 to 25% that of Wistar rat platelets. These results suggested that platelet abnormalities of TM rats are a typical characteristic of platelet SPD and are similar to those of FH rats, which are genetically different from TM rats. Therefore, TM rats may serve as a useful animal model for the study of platelet SPD.     

The proton gradient of secretory granules and glutamate transport in blood platelets during cholesterol depletion of the plasma membrane by methyl-β-cyclodextrin

Glutamate transport in blood platelets resembles that in brain nerve terminals because platelets contain neuronal Na⁺-dependent glutamate transporters, glutamate receptors in the plasma membrane, vesicular glutamate transporters in secretory granules, which use the proton gradient as a driving force, and can release glutamate during aggregation/activation. The acidification of secretory granules and glutamate transport were assessed during acute treatment of isolated platelets with cholesterol-depleting agent methyl-β-cyclodextrin (MβCD). Confocal imaging with the cholesterol-sensitive fluorescent dye filipin showed a quick reduction of cholesterol level in platelets. Using pH-sensitive fluorescent dye acridine orange, we demonstrated that the acidification of secretory granules of human and rabbit platelets was decreased by ∼15% and 51% after the addition of 5 and 15 mM MβCD, respectively. The enrichment of platelet plasma membrane with cholesterol by the application of complex MβCD-cholesterol (1:0.2) led to the additional accumulation of acridine orange in secretory granules indicating an increase in the proton pumping activity of vesicular H⁺-ATPase. MβCD did not evoke release of glutamate from platelets that was measured with glutamate dehydrogenase assay. Flow cytometric analysis did not reveal alterations in platelet size and granularity in the presence of MβCD. These data showed that the dissipation of the proton gradient of secretory granules rather than their exocytosis caused MβCD-evoked decrease in platelet acidification. Thus, the depletion of plasma membrane cholesterol in the presence of MβCD changed the functional state of platelets affecting storage capacity of secretory granules but did not evoke glutamate release from platelets.     

Hereditary types of thrombocytopenia with giant platelets and inclusion bodies in the leukocytes

Three forms of hereditary thrombocytopenia with giant platelets and inclusion bodies in the leukocytes have thus far been recognized. The May-Hegglin anomaly is characterized by giant platelets and spindle-shaped inclusion bodies in the leukocytes, which consist of 7-10 nm parallel-lying filaments. The Fechtner syndrome is a variant of the Alport syndrome, with inclusion bodies consisting of dispersed filaments, ribosomes and a few segments of rough and smooth endoplasmic reticulum. The Sebastian platelet syndrome shows the same platelet and leukocyte morphology observed in the Fechtner syndrome, but the additional anomalies e.g., the Alport syndrome, are lacking. The clinical signs and symptoms are variable. Most patients show only a mild bleeding tendency or are asymptomatic, but cases of severe postoperative hemorrhage have also been reported. Platelets can vary greatly in number, but are usually in the range of 20,000 to 120,000 platelets/microliters, showing a mean platelet volume of 15-20 fl, unimpaired in vitro function and, in addition to their size and unorganized microtubular system, normal morphology. To date, no platelet membrane defects have been defined. Because the megakaryocyte number and platelet kinetics are normal, the pathogenesis of thrombocytopenia in these giant platelet syndromes is unresolved; this is also true of the leukocyte inclusion bodies. Because of the ubiquity of electronic particle counters, asymptomatic patients are increasingly being identified, but they are often misdiagnosed as having autoimmune thrombocytopenia.     

Incorporation of a circulating protein into alpha granules of megakaryocytes

In order to determine whether or not proteins circulating in plasma can be incorporated into megakaryocytes and platelets, horseradish peroxidase (HRP) was injected intravenously into guinea pigs and these cells were examined for uptake by cytochemistry and electron microscopy. Enriched samples of megakaryocytes enabled ultrastructural analysis of large numbers of these rare bone marrow cells. In megakaryocytes, more than 50% of alpha granules contained HRP between 75 minutes and 7 hours after injection. At 24 hours, 25% of the megakaryocyte granules were peroxidase positive; less were so by 48 hours and none at 4 days. Thus, the findings demonstrate that a circulating protein can be endocytosed by megakaryocytes and rapidly packaged into alpha granules. A precipitous drop in circulating platelet numbers was observed 45 minutes after injection. At this time, circulating platelets showed the tracer only on the platelet plasma membrane, and none in platelet granules. Platelet numbers increased to 35% by 7 hours and only the platelet granules contained HRP. These platelets secreted the HRP stored in granules in response to thrombin. Unfortunately, our present studies do not allow us to distinguish between direct endocytosis by the platelet and/or shedding of new platelets from recently labeled megakaryocytes. Our studies are the first to demonstrate an endocytic pathway by which megakaryocytes can incorporate a circulating protein into alpha granules. An important physiologic implication of this endocytic pathway is the possible origin of certain alpha granule proteins from plasma.     

Signal transduction in normal and pathological thrombin-stimulated human platelets

Human blood platelets stimulated by thrombin undergo very rapid morphological changes, the most characteristic of which are pseudopod formation and granule centralization. These early changes in shape are accompanied by a transient decrease (30%) in phosphatidyl inositol 4,5-bisphosphate (PIP2) which occurs in the first 10 s after thrombin addition. Transient decreases in phosphatidyl inositol 4-phosphate (PIP) and phosphatidyl inositol (PI) occur later (20-30 s). These events lead to the formation of inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DG) and hence phosphatidate (PA). Two polypeptides are phosphorylated during the same time span: the myosin light chain (P20) and a 43 kDa protein (P43). Concomitant with these molecular changes, platelet 'release reaction' occurs, i.e., liberation of the different granule constituents into the external medium: the earliest concerns dense bodies which liberate adenine nucleotides, calcium and serotonin; alpha-granules then liberate adhesive and specific proteins and are followed by lysosomes which liberate hydrolases. Pathological platelets from patients with inherited disorders, presenting well-characterized and specific defects of either the platelet membrane (GT) or storage granules (GPS and HPS), have also been studied. The results obtained lead to the following conclusions: (1) the transducing system is normal in platelets unable to aggregate; (2) phosphorylation of P20 and P43 proteins can be complete with impaired release; and (3) when platelets lack alpha-granules the transducing system as well as the release of other granule populations are impaired. These results evidence the relationship between the absence of intraplatelet components and metabolic events.     

Identification of protein kinase Calpha as an essential, but not sufficient, cytosolic factor for Ca2+-induced alpha- and dense-core granule secretion in platelets

Upon activation, platelets release many active substances. Here, we have analyzed the mechanism governing Ca(2+)-induced secretion of von Willebrand factor stored in alpha-granules and 5-hydroxytryptamine in dense-core granules in permeabilized human platelets. Both secretions were dependent on ATP and cytosol. An essential factor for both granule secretions was purified from rat brain cytosol and identified to be protein kinase Calpha (PKCalpha) by partial amino acid sequencing. Purified PKCalpha efficiently stimulated both secretions in the presence of cytosol, whereas PKCalpha alone did not support the secretion of either type of granules, suggesting that PKCalpha is not a sufficient factor. Finally, in human platelet cytosol fractionated by a gel filtration column, the stimulatory activity for dense-core granule secretion paralleled with the concentration of PKC, suggesting that PKC could also be such a stimulatory factor in platelet cytosol. Thus, we identified PKCalpha as an essential, but not sufficient, cytosolic factor for the Ca(2+)-induced secretions of both alpha- and dense-core granules in platelets.     

Identification and characterization of LAMP-1 as an activation-dependent platelet surface glycoprotein

Platelets normally circulate in a quiescent state. When activated, they undergo biochemical and morphological changes which greatly alter their function and contribute to their role in thrombosis and hemostasis. We have identified, cloned, and sequenced a cDNA from a human unbilical vein endothelial cell library that encodes a 110-kDa integral membrane protein. This protein is present on the surface of activated but not resting platelets and has previously been identified as lysosomal-associated membrane protein 1 (LAMP-1). Half-maximal surface expression of platelet LAMP-1 was induced by concentrations of thrombin that resulted in lysosome enzyme release, not alpha-, or dense granule release. Also consistent with lysosome enzyme studies, there was little surface expression of LAMP-1 in response to the weak agonists ADP and epinephrine. In addition, sucrose density gradient fractionation of platelet granules showed colocalization of LAMP-1 with the lysosomal enzyme, beta-galactosidase, and not with markers of alpha- or dense granules. While we found virtually no LAMP-1 on the resting platelet surface (0-90 molecules/cell), we estimated a mean of 1175 LAMP-1 molecules on the thrombin-activated platelet surface. The translocation of this heavily glycosylated protein to the platelet surface upon stimulation may play a role in the adhesive, prothrombic nature of these cells.     

Localization of fibrinogen during ADP- or thrombin-induced aggregation of washed rabbit platelets

In contrast to human platelets, which aggregate poorly in response to ADP unless fibrinogen is present in the external medium, washed rabbit platelets form large aggregates in response to ADP without fibrinogen in the suspending medium. Addition of fibrinogen to the suspending medium of rabbit platelets frequently has little or no effect on the extent of ADP-induced platelet aggregation. We examined washed rabbit platelets by immunocytochemistry during ADP-induced aggregation and deaggregation and during thrombin-induced aggregation when the external medium did not contain added fibrinogen to determine if (a) fibrinogen was expressed on the surface of rabbit platelets that could support aggregation when the platelets were stimulated, or (b) fibrinogen secreted from the alpha granules supports platelet aggregation. Glutaraldehyde-fixed samples were prepared at different times after addition of ADP or thrombin, embedded in Lowicryl K4M, sectioned, incubated with sheep anti-rabbit fibrinogen, washed, reacted with gold-labeled anti-sheep IgG, and prepared for electron microscopy. The alpha granules of rabbit platelets were heavily labeled with immunogold; the platelet membrane was not labeled. During platelet aggregation and deaggregation in response to ADP, fibrinogen was not detectable on the platelet surface. In response to thrombin, large aggregates formed before fibrinogen was secreted from the alpha granules; fibrinogen was detectable focally at sites of granule discharge by 30-60 sec and fibrin formed by 3 min. Therefore, stimulated washed rabbit platelets can adhere to each other without large amounts of fibrinogen taking part in the close platelet-to-platelet contact, since aggregation occurs before detectable secretion, and large areas where the platelets are in contact are devoid of fibrinogen between the adherent membranes. Adhesion mechanisms not involving fibrinogen may support the aggregation of washed rabbit platelets.     

The ultrastructure of megathrombocytes in May-Hegglin's anomaly

A clinical and extensive electron microscopic study was made of the circulating granulocytes and thrombocytes from three female patients of a family with May-Hegglin anomaly. There have been observed inclusion bodies (D?hle-bodies) in a high proportion of polymorphonuclear leukocytes as well as a thrombocytopenia and giant platelets (so-called megathrombocytes). Other family members did not show hematologic abnormalities. The investigated giant platelets show ultrastructurally a different electron density of the alpha-granules, however, these structures present no typical formation of "bull's eye granules" which has been observed in other cases of May-Hegglin anomaly. Peripheral or central situated net-like structures of the endoplasmic reticulum have been found frequently. There were seen no microtubuli whereas glycogen particles were scattered within the peripheral cytoplasm.     

THE BIOSTIMULATORY EFFECT OF RED LASER IRRADIATION ON PIG BLOOD PLATELET FUNCTION

The molecular mechanisms of laser-induced changes in the cell structure and function are not well known. The authors examined the effects of low-power laser irradiation on unnucleated pig blood platelets. The obtained results showed that laser irradiation (1–5J) caused in blood platelets lipid peroxidation (measured as thiobarbituric acid reactive substances) and super-oxide anion generation, concomitant with the release of adenine nucleotides and proteins from platelets. The maximum platelet response to laser irradiation was observed when doses of 1.8–2J were used. Our results indicate that red laser irradiation induces: (1) platelet secretory process and the release of substances stored in the specific granules (adenine nucleotides, proteins); and (2) lipid peroxidation partly due to stimulation of endogenous arachidonate and production of its metabolites reacting with thiobarbituric acid.     

Human platelet dense granules contain polyphosphate and are similar to acidocalcisomes of bacteria and unicellular eukaryotes

Inorganic polyphosphate (polyP) has been identified and measured in human platelets. Millimolar levels (in terms of Pi residues) of short chain polyP were found. The presence of polyP of approximately 70-75 phosphate units was identified by 31P NMR and by urea-polyacrylamide gel electrophoresis of platelet extracts. An analysis of human platelet dense granules, purified using metrizamide gradient centrifugation, indicated that polyP was preferentially located in these organelles. This was confirmed by visualization of polyP in the dense granules using 4',6-diamidino-2-phenylindole and by its release together with pyrophosphate and serotonin upon thrombin stimulation of intact platelets. Dense granules were also shown to contain large amounts of calcium and potassium and both bafilomycin A1-sensitive ATPase and pyrophosphatase activities. In agreement with these results, when human platelets were loaded with the fluorescent calcium indicator Fura-2 acetoxymethyl ester to measure their intracellular Ca2+ concentration ([Ca2+]i), they were shown to possess a significant amount of Ca2+ stored in an acidic compartment. This was indicated by the following: 1) the increase in [Ca2+]i induced by nigericin, monensin, or the weak base, NH4Cl, in the nominal absence of extracellular Ca2 and 2) the effect of ionomycin, which could not take Ca2+ out of acidic organelles and was more effective after alkalinization of this compartment by the previous addition of nigericin, monensin, or NH4Cl. All of these characteristics of the platelet dense granules, together with their known acidity and high density (both by weight and by electron microscopy), are similar to those of acidocalcisomes (volutin granules, polyP bodies) of bacteria and unicellular eukaryotes. The results suggest that acidocalcisomes have been conserved during evolution from bacteria to humans.     

Morphology, origin and functions of the thrombocytes of Elasmobranchs

Thrombocytes of Torpedo marmorata Risso and Scyliorhynus stellaris L. (Elasmobranchs) are multiform from rounded to spindle-shaped. They originate in the spleen from prothrombocytes. They appear to be functionally correspondent to mammalian platelets because they form aggregates and adhere to glass and contain the same surface-connected canalicular system (SCCS) as in platelets, as proved by tannic acid treatment. Immunocytochemical staining have demonstrated, moreover, the presence in their cytoplasm of three platelet factors: platelet factor 4, beta-thromboglobulin and factor VIII related antigen. Stimulating agent, like collagen, ADP, noradrenaline, 5-hydroxytriptamine and thrombin induce aggregation of thrombocytes and the empting of their granules and vesicles containing very likely the platelet factors. The above observations leave few doubts on the functional equivalence of elasmobranch thrombocytes to mammalian platelets.