Microtubule coils in spread blood platelets

The fate of the circumferential bundle of microtubules in activated platelets has been a subject of disagreement. Thin sections of stimulated platelets fixed at multiple intervals following exposure to aggregating agents have revealed that the circumferential band is constricted into a tight ring around centrally concentrated organelles. However, studies of detergent-resistant platelet cytoskeletons fixed and either negatively stained or critical point dried after activation on polylysine-coated grids have revealed that microtubule rings disappear, leaving only fragments in the peripheral cytoplasm of spread cells. The present study has employed immunofluorescence on glass slides and the whole mount technique with detergent extraction and either negative staining or critical point drying to evaluate the fate of microtubules in surface-activated platelets treated with or without the microtubule stabilizing agent, taxol. Significant numbers of microtubule coils were visible in control and taxol-treated platelets stained indirectly with a fluorescein-coupled antibody to tubulin 30 to 60 minutes after surface activation on glass. Coils of microtubules were also visible in dendritic forms and in significant numbers of spread platelets on negatively stained or critical point dried whole mounts in the electron microscope. The findings support the concept that microtubule disassembly is not an integral step in early phases of platelet activation.     

Isolation of a 5-kilodalton actin-sequestering peptide from human blood platelets.

Resting human platelets contain approximately 0.3 mM unpolymerized actin. When freshly drawn and washed platelets are treated with saponin, 85-90% of the unpolymerized actin diffuses out. Analysis by polyacrylamide gel electrophoresis under nondenaturing conditions shows that the bulk of this unpolymerized actin migrates with a higher mobility than does pure G-actin, profilactin, or actin-gelsolin complex. When muscle G-actin is added to fresh or boiled saponin extract, the added muscle actin is shifted to the high-mobility form. The saponin extract contains an acidic peptide having a molecular mass in the range of 5 kDa, which has been purified to homogeneity by reverse-phase HPLC. This peptide also shifts muscle actin to the high-mobility form. Addition of either boiled saponin extract or the purified peptide to muscle G-actin also strongly and stoichiometrically inhibits salt-induced polymerization, as assayed by falling-ball viscometry and by sedimentation. We conclude that this peptide binds to the bulk of the unpolymerized actin in platelets and prevents it from polymerizing.     

Isolation of normal human megakaryocytes

S ummary . This paper reports a simple procedure for obtaining human megakaryocytes with a high purification and high recovery yield. Bone marrow cells, obtained from surgically removed ribs, were separated by a two-step procedure. Initially, a single cell suspension was enriched in megakaryocytes by equilibrium density centrifugation, the low density cell fraction was subsequently layered over a shallow continuous albumin gradient in a glass sedimentation chamber. Megakaryocytes averaged 0.03 ± 0.02% of all nucleated cells in the starting marrow cell suspension, after this procedure an average 80 ± 15% of the initial megakaryocyte population was recovered with a purity of 94 ± 4%. Previous methods, based upon the use of a two-step procedure, are reviewed. The theory of velocity sedimentation is discussed with regard to the differences in the methodology used, which account for the different results I obtained.     

Platelet-activating factor is a weak platelet agonist: Evidence from normal human platelets and platelets with congenital secretion defects

We have examined the effects of a novel platelet agonist, platelet activating factor (PAF), on human platelets. Irreversible aggregation and ¹⁴C-serotonin secretion in response to PAF (10^?5 M) was found to be dependent on both thromboxane production and secreted adenosine diphosphate (ADP). Liberation of arachidonic acid (AA) from membrane-bound phospholipids is a prerequisite step in platelet thromboxane production. Studies with ³H-AA-labeled platelets revealed that PAF (10^?5 M) was a weak stimulus for the mobilization of AA. In addition, PAF (10^?5M) was found to be a weak inducer of thromboxane synthesis (mean = 6 pmol/10⁸ platelets) as compared to thrombin 5 U/ml (mean = 177 pmol/10⁸ platelets), measured using a radioimmunoassay for thromboxane B₂. Formation of phosphatidic acid is an early step in stimulus-response coupling in platelets. Our studies indicate that PAF is a weak stimulus for phosphatidic acid formation as well. To obtain further insights into its action, we examined the effect of PAF on platelets from three groups of patients with congenital secretion defects: patients with the storage pool deficiency, those with impaired thromboxane synthesis due to impaired liberation of AA from phospholipids, and those with impaired secretion despite normal granule stores and thromboxane production. The response to PAF was impaired in all patients, providing further evidence that PAF-induced platelet activation is dependent on secreted ADP and thromboxane A₂ synthesis, and occurs by mechanisms common to a number of agonists. Overall, these studies indicate that PAF is a weak platelet agonist.     

Isolation and structural characterization of the polypeptide subunits of membrane glycoprotein IIb-IIIa from human platelets

We have previously demonstrated the isolation of platelet membrane glycoprotein IIb-IIIa by affinity chromatography with a specific monoclonal antibody. We have now separated the polypeptide subunits IIb and IIIa of the isolated glycoprotein by preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis and have compared their structural features. Both IIb and IIIa contain approximately 15% carbohydrate, but IIIa contains a larger percentage of mannose residues, suggesting the presence of high mannose as well as complex N- linked oligosaccharide chains. The amino acid compositions are sufficiently similar to imply areas of sequence homology between the two subunits. To examine further the relationship between the subunits, we digested a mixture of 125I-IIb and 131I-IIIa with trypsin and then separated the radiolabeled peptides by high performance liquid chromatography. The resultant peptide maps of IIb and IIIa are completely different. This indicates that neither subunit is derived from the other and suggests that polypeptides IIb and IIIa are products of separate genes.     

Isolation of circumferential microtubules from platelets without simultaneous fixation

Circumferential bands of microtubules (MT) support the discoid shape of resting platelets and participate with the contractile apparatus in shape change and internal contraction following activation. Elucidation of interactions between the circumferential coils and proteins of the stable and contractile cytoskeleton is essential for understanding MT function in platelet physiology. A previous investigation demonstrated that the circumferential rings can be isolated intact from resting platelets following simultaneous exposure to glutaraldehyde and Triton X-100. However, the use of fixation prevented the characterization of protein interactions. The present study has circumvented this problem by developing a procedure for isolating intact microtubule coils from detergent-treated platelets without the use of fixative agents. Incubation of the platelets for intervals of 30 to 60 minutes with the microtubule-stabilizing agent taxol preserved the circumferential bundle after extraction with Triton X-100 even after washing five times. The procedure has made it possible to carry out protein studies on isolated microtubule rings and associated proteins.     

Apolipoprotein B release from activated human platelets

Apolipoprotein B (apoB) release from activated washed human platelets was measured by enzyme-linked immunosorbent assay (ELISA) using monospecific rabbit antibodies to human low density lipoprotein (LDL). Activation of platelets with thrombin, Ca2+-ionophore A23187 or stable analogue of prostaglandin endoperoxides U46619 stimulated release of approximately 20 ng apoB/10(8) platelets. Thrombin-induced apoB release was inhibited by the prostacyclin analogue carbacyclin. Dose-response curves of thrombin stimulation and carbacyclin inhibition of apoB and beta-thromboglobulin (beta-TG) release were very similar. Treatment of platelets with heparin did not remove significant amounts of apoB or affect the subsequent release of apoB induced by thrombin. The results of density gradient ultracentrifugation indicated that most of the apoB was released in the LDL density range. These data suggest that human platelets contain immunoreactive apoB, which can be released during platelet activation.     

Factor-V expression in platelets from human megakaryocytic culture

The origin of platelet-factor-V has long been discussed. To elucidate whether and when human platelet-factor-V is synthesized by megakaryocytes, we utilized in vitro-generated megakaryocytes capable of producing platelets. Factor-V gene was silent in purified progenitors and megakaryocytic precursors but was expressed in late culture phase and maintained also in platelets. Similarly, factor-V protein was expressed in mature proplatelet-bearing megakaryocytes (immunofluorescence analysis); it was also detectable in cultured megakaryocytes and platelets (Western blotting) and within permeabilized cultured platelets (flow cytometry). The absence of other cells in our culture system indicates conclusively that human megakaryocytes synthesize factor-V.     

Isolation of megakaryocytes from human placentae

Shedding of cytoplasm from circulating megakaryocytes (MKs) within the pulmonary vasculature suggests the lungs are an important site for normal platelet production. Fetal lungs receive only a minor fraction of the circulating blood volume. The placenta may act as a site for intrauterine platelet formation. Isolation of MKs from fetal vessels within the placenta has not been previously reported. Immediately after delivery, 3 human placentae were subjected to forward and retrograde perfusion across the placental capillary bed on the fetal side. MKs in perfusates were harvested by 'whole blood filtration' and identified by morphological and immunochemical methods. All perfusates yielded MKs. Qualitatively MKs with copious cytoplasm were more commonly found in perfusates collected from fetal arteries compared with those from fetal veins. This is consistent with filtration of MKs and fragmentation of their cytoplasm within the placental microcirculation to produce platelets. Perfusion of human placentae followed by filtration of perfusates is a useful technique for harvesting fetal MKs and permitting further elucidation of their physiological role.     

Characterization of platelets from normal mink and mink with the Chediak-Higashi syndrome

Bleeding times of mink with the Chediak-Higashi (CH) syndrome was markedly prolonged. Platelet counts were normal but there was an impaired platelet aggregation response to collagen. The metabolic adenine nucleotide pool of platelets from normal and CH mink was labeled with 14C-adenine and the platelets were gel-filtered. Gel-filtered platelets (GFP) from CH mink contained only 37.9% of the adenosine triphosphate (ATP) and 9.6% of the adenosine diphosphate (ADP) found in normal platelets and the ATP/ADP ratio was similar to the 14C-ATP/14C-ADP ratio. Platelet content of Ca2+, Mg2+, and in particular 5-hydroxytryptamine was decreased. When GFP were incubated with thrombin to induce maximal secretion, only negligible amounts of ATP and ADP were released. The specific activity of the extracellular nucleotides approximated that within the platelet. These findings suggest that the stored nucleotide pool in CH platelets is virtually absent and that the abnormalities in platelet function may be due, in part, to the essential absence of secretable ADP and serotonin. The release of Ca2+ and Mg2+ by CH platelets was 56% and 27.8% of normal, respectively.