Bovine Platelets Research Articles

Assembly and GPIIIa Content of Cytoskeletal Core in Platelets Agglutinated With Bovine von Willebrand Factor

The association between occupancy of the von Willebrand factor (vWf) receptor glycoprotein (GP) Ib, agglutination, and the assembly and composition of the cytoskeletal core was studied in 125I-surface-labeled aspirin-treated washed platelets. Binding of ligands to GPIIb-IIIa and platelet aggregation were abolished by addition of EDTA. Platelet agglutination induced by bovine vWf generated a complete cytoskeletal core (Triton-insoluble residue), shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to be composed of actin-binding protein (ABP) (260 Kd), 235-Kd protein, myosin heavy chain (200 Kd), alpha-actinin (100 Kd), and actin (43 Kd). In addition, autoradiography of the gels showed a 125I 105-Kd GP, identified by immunoblot as GPIIIa, as well as GPIb, GPIIb, and another band at 87 Kd, probably GPIV. Neither cytoskeletal assembly nor GPIIa incorporation was altered if calpain was inhibited with leupeptin. Platelet suspensions exposed to bovine vWf without stirring (ie, nonagglutinated) or platelets in which agglutination was inhibited with ADP showed smaller cytoskeletons with little ABP, 235 Kd protein, and alpha-actinin. Autoradiographs showed mainly GPIb. Cytochalasin D (CD) and monobromobimane (MB) enhanced agglutination and prevented the inhibitory action of ADP on bovine vWf-induced platelet agglutination. CD markedly inhibited the assembly of the cytoskeletal core as well as GPIIIa retention, whereas MB resulted in a large Triton-insoluble residue which contained GPIIIa. Thus, development of a platelet cytoskeletal core is apparently not required for agglutination, but when a cytoskeletal core is assembled in agglutinated platelets, GPIIIa is retained.

Activation of soluble guanylate cyclase by nitrovasodilators is inhibited by oxidized low-density lipoprotein

In the presence of oxidized low-density lipoprotein the stimulatory effects of nitric oxide, sodium nitroprusside and S-nitrosoglutathione on soluble guanylate cyclase partially purified from bovine platelets were diminished in a concentration-dependent manner with IC 50 values around 100 μg total cholesterol/ml. This inhibitory effect was potentiated about 10-fold when the enzyme was preincubated with the lipoprotein for 10 minutes at 37 °C which indicates a direct interaction of the lipoprotein with the guanylate cyclase. As oxidized low-density lipoprotein is present in the wall of atherosclerotic arteries, we suggest that the impaired response of atherosclerotic blood vessels to vasodilators may be due to a diminished activation of smooth muscle guanylate cyclase.

Comparative effects of trichothecene mycotoxins on bovine platelet function: Acetyl T-2 toxin, a more potent inhibitor than T-2 toxin

The effects of the trichothecene mycotoxins (acetyl T-2 toxin, T-2 toxin, HT-2 toxin, palmityl T-2 toxin, diacetoxyscirpenol (DAS), deoxynivalenol (DON), and T-2 tetraol) on bovine platelet function were examined in homologous plasma stimulated with platelet activating factor (PAF). The mycotoxins inhibited platelet function with the following order of potency: acetyl T-2 toxin > palmityl T-2 toxin = DAS > HT-2 toxin = T-2 toxin. While T-2 tetraol was completely ineffective as an inhibitor, DON exhibited minimal inhibitory activity at concentrations above 10×10(-4)M. The stability of the platelet aggregates formed was significantly reduced in all mycotoxin treated platelets compared to that of the untreated PAF controls. It is suggested that the increased sensitivity of PAF stimulated bovine platelets to the more lipophilic mycotoxins may be related to their more efficient partitioning into the platelet membrane compared to the more hydrophilic compounds.

ESR analysis with long-chain alkyl spin labels in bovine blood platelets. Relationship between the increase in membrane fluidity by alcohols and phenolic compounds and their inhibitory effects on aggregation

Four spin-labeled probes (5-doxylstearic acid (5-NS), its methyl ester (5-NMS), 16-doxylmethylstearate (16-NMS) and 4-( N, N-dimethyl- N-pentadecy)ammonium-2,2,6,6-tetramethylpiperidine-1-oxyl (CAT-15)) were used to monitor membrane fluidity change in bovine platelets induced by three alkyl alcohols, benzyl alcohol and two phenolic compounds. The relationship between the increase in membrane fluidity induced by these compounds and their inhibitory effects on platelet aggregation was observed. Experiments with the four probes showed that n-hexyl alcohol induced decreases in the order parameter of 5-NS and apparent rotational correlation times of the other probes at the same minimal alcohol concentration. The decreases were observed in the concentration range that inhibited aggregation. n-Amyl alcohol and n-butyl alcohol decreased the values of the parameters of the above mentioned only at higher concentrations that were dependent on their hydrophobicities. Like alkyl alcohols, benzyl alcohol and phenolic compounds decreased the values of the parameters in the concentration ranges in which these compounds inhibited platelet aggregation. The concentration of these compounds causing 50% inhibition of platelet aggregation, the IC 50 values, and data on 5-NS-labeled platelets indicated that they inhibited aggregation and decreased the value of the order parameter at lower concentrations relative to their P oct values in comparison to the effective concentrations of alcohols. Phenolic compounds also decreased the values of the apparent rotational correlation times of 5-NMS and 16-NMS. These results indicate that the inhibition of platelet aggregation by alcohols and phenolic compounds is due to membrane perturbation in wide range in depths within the lipid bilayer.

Repression of Serotonin Secretion by an Endogenous Ca2+ Protease in Electropermeabilized Bovine Platelets1

Micromolar levels of free calcium ions added to the extracellular medium elicit secretion of serotonin from electropermeabilized bovine platelets in the presence of millimolar levels of Mg-ATP. Such Ca2(+)-dependent secretion of serotonin was almost completely impaired when the permeabilized platelets were preincubated for 1 min at 35 degrees C in 100 microM Ca2+ without Mg-ATP. The half-maximal effect was observed with about 45 microM Ca2+ in the preincubation medium. Inhibitors of serine-thiol protease, such as leupeptin and antipain, suppressed the impairment of the secretion of serotonin by the preincubation with Ca2+. Electron microscopic observation revealed that disorganization of the cytoskeletal structures, in particular of the membrane undercoat and the network of microfilaments, accompanied the impairment of secretion of serotonin. Microfilaments were also found to be dissociated from dense granules that contained serotonin. These morphological changes were also suppressed when antipain was included in the Ca2(+)-preincubation medium. Coincident with these morphological changes, the following biochemical changes were observed in 100 microM Ca2+ but not in the presence of Ca2+ and antipain. The amount of Triton-insoluble cytoskeleton and the acto-myosin content of the dense-granule fraction were markedly decreased. The decrease in Triton-insoluble cytoskeletons was quantitatively correlated with the degree of impairment of secretion of serotonin. Immunoblot analysis of EGTA extracts of the cells showed that the 240-kDa spectrin in platelets was degraded to a 235-kDa fragment, and a 260-kDa actin-binding protein (ABP) in platelets was partially degraded to 190- and 110-kDa components.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibitory mechanism of cis-polyunsaturated fatty acids on platelet aggregation: the relation with their effects on Ca 2+ mobilization, cyclic AMP levels and membrane fluidity

The in vitro inhibitory effects of cis-polyunsaturated fatty acids, linolenic (18:2 Δ 9,12), α-linoleic (18:3 Δ 9,12,15) and eicosatrienoic (20:3 Δ 11,14,17) acid, on bovine platelet aggregation and their inhibitory mechanism were investigated. These fatty acids inhibited platelet aggregation induced by ADP and thrombin to similar extent. Fluorescence analyses with fura-2-loaded platelets showed that, in the concentration ranges that inhibited aggregation, they also inhibited agonist-induced increase in cytoplasmic Ca 2+. According to radioimmunoassay study, addition of these fatty acids increased cyclic AMP contents in the presence of theophylline corresponded with their inhibitory effects on aggregation. These fatty acids induced a 1.6–1.8-fold increase over basal concentration of cyclic AMP in the concentration ranges that fully inhibited aggregation. On the other hand, saturated fatty acid, stearic acid, affected neither aggregation nor cyclic AMP levels. As reported previously ((1985) Biochim. Biophys. Acta 818, 391), these unsaturated fatty acids induced increase in membrane fluidity in the same concentration range. These results suggest that inhibition of platelet aggregation by cis-polyunsaturated fatty acids is due to the increase in cyclic AMP levels. This increase seems to be due to stimulation of adenylate cyclase which is mediated by membrane perturbation.

Cooperativity between platelet-activating factor and collagen in aggregation of bovine platelets III

In cooperative aggregation of bovine platelets induced by simultaneous addition of PAF and collagen at subthreshold concentrations the following observations were made. (i) Formation of phosphatidic acid and arachidonic acid metabolites, which characterize PAF and collagen alone-induced aggregation, respectively, was observed very obviously. (ii) A thromboxane antagonist did not completely block the cooperative aggregation. The extent of residual aggregation activity was dependent on concentration of collagen used in the simultaneous administration with PAF. These results suggest that both signal transduction pathways activated by PAF and collagen alone at high concentrations are attained by simultaneous addition of both agonists at subthreshold concentrations through unknown mechanisms.

Anchorage of secretion-competent dense granules on the plasma membrane of bovine platelets in the absence of secretory stimulation.

The ultrastructural changes in electropermeabilized bovine platelets that accompany the Ca2(+)-induced secretion of serotonin were investigated in ultra-thin sections of chemically fixed cells. Such preparations permitted us to study both the localization of and the structures associated with serotonin-containing dense granules. Localization of dense granules within cells was examined by measuring the shortest distances between the granular membranes and the plasma membrane. About 40% of total granules were located close to the plasma membrane at an average distance of 10.8 +/- 1.6 nm. 71% of the total number of granules were localized at a similar average distance of 12.5 +/- 2.7 nm in intact platelets. The percentage of granules apposed to the plasma membrane corresponded closely to the percentage of total serotonin that was maximally secreted after stimulation of the permeabilized (38 +/- 4.9%) and the intact platelets (72 +/- 3.6%). Furthermore, the percentage of granules anchored to the membrane, but not of those in other regions of permeabilized cells, decreased markedly when cells were stimulated for 30 s by extracellularly added Ca2+. The decrease in the numbers of granules in the vicinity of the plasma membrane corresponded to approximately 22% of the total number of dense granules that were used for measurements of the distances between the two membranes and corresponded roughly to the overall decrease (15%) in the average number of the granules per cell. Most dense granules were found to be associated with meshwork structures of microfilaments. Upon secretory stimulation, nonfilamentous, amorphous structures found between the plasma membrane and the apposed granules formed a bridge-like structure that connected both membranes without any obvious accompanying changes in the microfilament structures. These results suggest that the dense granules that are susceptible to secretory stimulation are anchored to the plasma membrane before stimulation, and that the formation of the bridge-like structure may participate in the Ca2(+)-regulated exocytosis.

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.

Feedback regulation of platelet function by 12 S-hydroxyeicosatetraenoic acid: Inhibition of arachidonic acid liberation from phospholipids

We have proposed a mechanism that platelet aggregation is regulated by its 12-lipoxygenase product, 12 S-hydroxyeicosatetraenoic acid (12-HETE) (Sekiya, F., Takagi, J. and Saito, Y. (1989) Thrombos. Res. 56, 407–415). Inhibition of endogenous 12-HETE production by 15-HETE, a specific inhibitor of 12-lipoxygenase, accelerated aggregation of bovine platelets in response to collagen and arachidonic acid liberation from phospholipids was enhanced. Exogenously added 12-HETE suppressed collagen-induced liberation of arachidonic acid and the aggregation was also inhibited. On the other hand, 12-HETE did not interfere with thromboxane synthesis from free arachidonic acid in a cell-free system. These observations suggest that 12-HETE exerts a negative feedback to prevent excess aggregation through interference with arachidonic acid liberation from membrane phospholipids.

Cooperativity between platelet-activating factor and collagen in aggregation of bovine platelets, II

When subthreshold amounts of platelet-activating factor (PAF) and collagen were added simultaneously, strong aggregation of platelets was induced. However, each agonist alone at these concentrations could not induce aggregation at all (S. Kojima et al , (1987) Biochem. Biophys. Res. Commun. 145, 915–920). This cooperativity was observed not only in aggregation but also in changes of intracellular Ca 2+ concentration, 47 kDa protein phosphorylation, and formation of thromboxane. These findings suggest that various steps of signal transduction pathways are enhanced during their cooperativity.

Phosphorylation of bovine platelet myosin by protein kinase C

Bovine platelet myosin is phosphorylated by protein kinase C at multiple sites. Most of the phosphate is incorporated in the 20,000-dalton light chain although some phosphate is incorporated in the heavy chain. Phosphorylation of the 20,000-dalton light chain of platelet myosin is 10 times faster than the phosphorylation of smooth muscle myosin. Platelet myosin light chain is first phosphorylated at a threonine residue followed by a serine residue. Dominant phosphorylation sites of the 20,000-dalton light chain are estimated as serine-1, serine-2, and threonine-9. Prolonged phosphorylation by protein kinase C resulted in an additional phosphorylation site which, on the basis of limited proteolysis, appears to be either serine-19 or threonine-18. Phosphorylation by protein kinase C causes an inhibition of actin-activated ATPase activity of platelet myosin prephosphorylated by myosin light chain kinase. Inhibition of ATPase activity is due to a decreased affinity of myosin for actin, and no change in Vmax is observed. It is shown that platelet myosin also exhibits the 6S to 10S conformation transition as judged by viscosity and gel filtration methods. Mg2(+)-ATPase activity of platelet myosin is paralleled with the 10S-6S transition. Phosphorylation by protein kinase C affects neither the 10S-6S transition nor the myosin filament formation. Therefore, the inhibition of actin-activated ATPase activity of platelet myosin is not due to the change in the myosin conformation.

Actin cytoskeleton of resting bovine platelets

Actin filaments in resting discoid bovine platelets were examined by fluorescence and electron microscopy. Rhodamine-phalloidin staining patterns showed a characteristic wheel-like structure which consisted of a central small circle connected by several radial spokes to a large peripheral circle. This wheel-like structure was composed of actin filaments forming a characteristic arrowhead structure with heavy meromyosin from muscle. Actin filaments were densely arrayed in parallel with a marginal microtubule band and radiated out from the center to the periphery. Platelets treated with colchicine lost their marginal microtubule band but retained their wheel-like structure and normal discoid form. Cytochalasin B disrupted the wheel-like structure but not the marginal microtubule band or the normal discoid form. After simultaneous treatment with both cytochalasin B and colchicine, platelets lost their discoid shape. These results suggest that actin filaments and microtubules both play important roles in the maintenance of the discoid shape of resting bovine platelets.

Effects of hypomagnesemia on reactivity of bovine and ovine platelets: possible relevance to infantile apnea and sudden infant death syndrome.

Blood platelet function and possible involvement in death of hypomagnesemic ruminants was investigated with 26 Angus cows, 15 mature Hampshire wethers, eight Finnish-Hampshire ewes, and 36 growing Dorset lambs. Hypomagnesemia was induced by feeding vegetative spring tall fescue to 13 cows and semipurified diets low in Mg to nine wethers, four ewes, and 18 lambs. In comparison with controls, dietary treatments reduced plasma Mg concentrations 55% in cows, 36% in wethers, 66% in ewes, and 78% in lambs. Hypomagnesemia reduced in vitro reactivity of cow and lamb platelets to thrombin, ADP, and platelet active collagen, but in vitro tests may not accurately reflect in vivo platelet reactivity. Microscopic examination of platelet-rich plasma revealed a threefold increase in clumped platelets from four hypomagnesemic ewes compared to four normomagnesemic ewes. This suggests that in vivo activation and exhaustion of platelets may have contributed to reduced in vitro platelet reactivity. Six of 18 hypomagnesemic lambs died spontaneously in tetany after 2-12 months on low-Mg diets. Heart and lung lesions were markedly similar to pathological changes induced in other lambs by intravascular activation of platelets with 500 micrograms of vascular collagen fibrils per kg body weight injected intravenously. These results suggest the possibility of abnormal blood platelet activation as a significant mortality risk factor in severe hypomagnesemia.

Demonstration of Secondary Lysosomes in Bovine Megakaryocytes and Platelets Using Acid Phosphatase Cytochemistry with Cerium as a Trapping Agent

The ultrastructure of lysosomes from bovine megakaryocytes (MK) and platelets was characterized using acid phosphatase cytochemistry with beta-glycerophosphate as substrate and cerium as a trapping agent. The technique was easily reproducible; cerium-phosphate precipitates were uniform, readily visualized, and there was a virtual absence of nonspecific reaction product. Acid phosphatase was localized in the trans aspect of the Golgi complex and/or granules of less than 50 nm to 650 nm diameters in MK at all stages of maturation. Forty percent of the MK lysosomes contained inclusions of variable shapes, sizes and electron-density and were classified as secondary lysosomes. Twenty-four percent of the platelet sections contained acid phosphatase-positive granules. Fifty-four percent of these were secondary lysosomes. This is the initial report demonstrating secondary lysosomes in either resting MK or platelets using acid phosphatase cytochemistry. These findings suggest that MK and platelet lysosomes have an intracellular function in resting MK and platelets.

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.

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.

Elucidation of a role of plasma albumin during collagen-induced aggregation of bovine platelets

Bovine platelets of which phospholipids were labeled with [ 14C] arachidonate were stimulated by collagen. Omitting albumin in suspending medium suppressed aggregation and arachidonate liberation from phospholipids. Analysis of [ 14C] arachidonate metabolites released into medium revealed that release of 12 S -hydroxyeicosatetraenoic acid (12-HETE) from stimulated platelets was dependent on the existence of albumin, while thromboxane B 2 release was not. Moreover, exogenously added 12-HETE inhibited collagen-induced aggregation. These observations suggest that albumin potentiates aggregation by preventing intracellular accumulation of 12-HETE, and strengthen our previous finding that albumin in plasma is very essential for collagen-induced aggregation of platelets (Sekiya, F., Takagi, J., Kasahara, K., Inada, Y. and Saito, Y. (1988) Thrombos. Res. 50, 837–846.).

Phosphorylation of a second site for myosin light chain kinase on platelet myosin

The 20,000-dalton light chain of bovine platelet myosin is phosphorylated at two sites by myosin light chain kinase. The first and second phosphorylation sites are at a serine and a threonine residue, respectively. The location of the phosphorylation sites was determined by using limited proteolysis. The N-terminal sequence of the 17,000-dalton tryptic fragment of platelet myosin 20,000-dalton light chain was found to be identical with that of gizzard 20,000-dalton light chain from Ala-17 to Phe-33. On the basis of these results and the distribution of 32P among the proteolytic fragments, it was concluded that serine-19 and threonine-18 were the two phosphorylation sites. Phosphorylation at the threonine residue markedly increases the actin-activated ATPase activity of myosin. It was found that platelet myosin forms 10S and 6S conformations and its Mg2+-ATPase activity parallels the transition from the 6S to the 10S conformation. The conformational transition was influenced by phosphorylation at both sites, and the phosphorylation at the threonine residue further shifted the equilibrium toward the 6S conformation. The phosphorylation at the threonine residue also induced thick filament formation in the presence of ATP. These results suggest that the phosphorylation at the threonine residue as well as at the serine residue may play an important role in the contractility of nonmuscle cells.

A Comparison of the Antiplatelet Effect of Aspirin on Human and Bovine Platelets

The risk of thromboembolism in human patients or animal models with blood contacting prosthetic devices is well documented. Aspirin is used frequently as an antiplatelet agent to minimize this risk. Although the inhibitory effect of aspirin on human platelets has been clearly established, preliminary studies to examine this effect on bovine platelets revealed a minimal inhibition of platelet function in vivo as well as in vitro. Because a considerable amount of implant research is carried out in bovine models, it was considered important to evaluate carefully the antiplatelet effect of aspirin on bovine platelets. To evaluate the effect of aspirin, experiments were conducted on human and bovine blood in vitro as well as after the administration of aspirin up to 4,000 mg/day (p.o.) in calves and 2,500 mg/day in humans. Appropriate amounts of buffered aspirin were added to heparinized or citrated blood incubated for 25 min and centrifuged to obtain platelet rich plasma (PRP). An aliquot of PRP was then challenged by a predetermined concentration of ADP (an aggregating agent) or collagen to evaluate platelet aggregation and release reaction. After aspirin was administered in vivo, blood was drawn from the animal at predetermined intervals to evaluate platelet function. In human blood, the inhibitory effect of aspirin was discernable at 80 mg/L in vitro and 2,500 mg/day when ingested orally. Under identical experimental conditions, no inhibition of bovine platelet aggregation was observed using dosages of aspirin up to 1,000 mg/L in vitro or 4,000 mg/day in vivo. These observations suggest that although the antiplatelet effect of aspirin is quite pronounced in humans, bovine platelets do not show a similar response to this drug. This should be an important consideration when platelet inhibition is desired during postoperative management of calves.