Biosynthesis and secretion of beta-thromboglobulin by a human megakaryoblastic cell line (MEG-01).

The de-novo synthesis and secretion of beta-thromboglobulin (BTG) by a human megakaryoblastic cell line (MEG-01) were studied by measuring and immunoblotting of BTG in culture supernatant and immunoprecipitation of radiolabeled BTG synthesized after incubation with [35S]methionine. It was demonstrated that BTG synthesized by MEG-01 was secreted into culture media in a monomer form having a molecular weight of 8,800. Furthermore, we purified BTG from culture medium of MEG-01 with a heparin affinity column and compared BTG from MEG-01 with that from normal platelets. The molecular weights of BTG purified from both sources were 8,800 using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). These results provide direct evidence for the synthesis and secretion of BTG by megakaryocytes.

Inhibitory effect of highly purified human platelet beta-thromboglobulin on in vitro human megakaryocyte colony formation.

The effect of highly purified human beta-thromboglobulin (beta TG), a glycoprotein present in platelet alpha granules, was tested on human bone marrow in vitro megakaryocyte (MK) colony formation. A concentration of 5 micrograms/ml of beta TG induced a 50% reduction in the number of MK colonies, and concentrations of 10 and 20 micrograms/ml completely inhibited MK growth. This inhibition was of importance for MKs because a higher concentration of beta TG (10 micrograms/ml) was needed to obtain a nonsignificant decrease in erythroblastic progenitors (erythroid burst-forming units, BFU-E), and no inhibition of granulocyte-macrophage colony-forming units (CFU-GM) was observed. beta TG acts mainly on maturation of MKs. These results indicate that beta TG could play a role in the physiological regulation of platelet production by MKs.

The neutrophil-activating proteins interleukin 8 and beta-thromboglobulin: in vitro and in vivo comparison of NH2-terminally processed forms.

Isolation of the human neutrophil activating protein (NAP) interleukin 8 (IL8) from leukocytes has revealed that it is structurally related to beta-thromboglobulin (beta TG) from platelets. Both these proteins occur as natural mixtures of multiple forms, differing from each other by unequal truncation at the NH2 terminus. In this study we have compared IL8 and beta TG forms for in vitro and in vivo neutrophil activation. In contrast to IL8, none of the beta TG forms were found to exert granulocyte chemotactic activity in vitro, as measured in the agarose assay. However, fractions rich in the most extensively processed forms of beta TG (e.g. NAP-2) as well as pure NAP-2 did induce lactoferrin release from granulocytes, whereas fractions containing only the longer forms (e.g. connective tissue-activating peptide III) were inactive. In order to observe this in vitro effect, about 10-fold less IL8 (10 nM) than NAP-2 was required. In the presence of a vasodilator substance low doses (2-20 pmol) of IL8 and the shorter forms of beta TG caused granulocyte accumulation and plasma leakage in rabbit skin whereas the longer forms of beta TG again failed to do so. Finally, granulocytosis induction following i.v. injection was found to occur with NAP-2. At the maximal dose tested (250 pmol), this in vivo effect of NAP-2 was less pronounced than that of IL8. In the case of IL8, NH2-terminal processing did not seem to affect granulocyte stimulatory activity. It should be noted, however, that the extent of processing of IL8 is less than that occurring with beta TG. It can be concluded that the platelet factor beta TG, structurally related to the monokine IL8, can also play a role in neutrophil activation during inflammatory reactions.

Dose-dependent effects of verapamil and nifedipine on in vivo platelet function in normal volunteers.

The effects of 1 week of treatment with low and moderate doses of verapamil or nifedipine upon platelet function has been studied in 12 healthy volunteers. The ex vivo platelet aggregation threshold for ADP or adrenaline was not altered by verapamil or nifedipine. The plasma concentrations of beta-thromboglobulin and platelet factor 4 were significantly reduced by low but not by moderate doses of verapamil and nifedipine. Low doses of verapamil and nifedipine inhibit in vivo platelet activity in healthy volunteers.

Purification of granulocyte chemotactic peptide/interleukin-8 reveals N-terminal sequence heterogeneity similar to that of beta-thromboglobulin.

Stimulated human peripheral blood leukocytes produce a chemotactic factor for granulocytes (granulocyte chemotactic peptide/interleukin-8; GCP/IL-8), which is structurally related to platelet-derived beta-thromboglobulin. Analytically pure CGP/IL-8 and beta-thromboglobulin could be obtained after three purification steps, comprising adsorption to silicic acid, heparin-Sepharose chromatography and ion-exchange chromatography. Although GCP/IL-8 and beta-thromboglobulin had a similar affinity for heparin, they could be separated on a cation-exchange column. Both molecules were heterogeneous in that 6-7-kDa protein doublets were detected upon SDS/PAGE. N-terminal amino acid sequence analysis revealed the presence of six immunologically related but differently truncated polypeptides of beta-thromboglobulin, of which only two corresponded to previously described forms. Similarly, apart from a major polypeptide, five minor species of GCP/IL-8 were detected that also differed by N-terminal truncation. The most processed forms of beta-thromboglobulin and GCP/IL-8 were found to have their N-terminus in that region of the primary structure where a significant similarity between the two molecules starts. GCP/IL-8 was found to be chemotactic for granulocytes with a specific activity of 10(5) units/mg, whereas none of the beta-thromboglobulin species possessed detectable chemotactic activity.

Isolation of bovine platelet cationic proteins which inhibit the surface-mediated activation of factor XII and prekallikrein.

A possible role of bovine platelets in the surface-mediated activation of Factor XII and prekallikrein was studied, using the contact system reconstituted with the purified proteins from bovine plasma. The washed platelets before and after aggregation by ADP, thrombin or collagen did not show any ability to trigger or accelerate the activation of Factor XII and prekallikrein. On the contrary, these aggregates showed a potent inhibitory activity on the activation of those zymogens triggered by kaolin, amylose sulfate and sulfatide. The inhibitory substances from the supernatant of the thrombin-induced aggregates were separated into two major fractions, a low affinity fraction and a high affinity fraction, on a heparin-Sepharose column. The high affinity protein was identified as platelet factor 4, based on the amino acid composition. From the low affinity fraction, a beta-thromboglobulin (beta-TG)-like substance and three kinds of unknown proteins, named LA1, LA2, and LA3, were isolated by gel-filtration on a column of Sephadex G-100 or Sephadex G-75 followed by chromatography on a column of Mono S. The molecular weights of LA1, LA2, and LA3 were estimated to be 35,000, 26,000, and 11,000, respectively, on SDS-PAGE. LA2 was identified as a carbohydrate-less LA1, as judged from the amino acid composition and carbohydrate content. The inhibitory activities of these five cationic proteins on the activation of Factor XII and prekallikrein mediated with amylose sulfate, sulfatide and kaolin were different from each other. In the case of kaolin-mediated activation, LA3 was the most potent inhibitor, while platelet factor 4 and beta-TG-like substance did not show any significant inhibitory activity. Moreover, the inhibitory activities of all the cationic proteins were not correlated with their anti-heparin activities. Since these proteins were rapidly liberated from platelets by the action of the stimulants, the present results demonstrate a negative role of platelets in the surface-mediated activation of Factor XII and prekallikrein.

Identification and separation of secreted platelet proteins by isoelectric focusing. Evidence that low-affinity platelet factor 4 is converted to beta-thromboglobulin by limited proteolysis.

Low-affinity platelet factor 4 and beta-thromboglobulin are low molecular weight platelet secretory proteins that have common antigenic determinants. Four amino acids (Asn-Leu-Ala-Lys) at the amino terminus of beta-thromboglobulin are deleted, but the remaining sequences of the two peptides appear to be identical. Low-affinity platelet factor 4 and beta-thromboglobulin have respective isoelectric points at pH 8.0 and at pH 7.0. Identification, quantitation, and separation of both proteins was achieved by a method combining preparative isoelectric focusing and specific radioimmunoassay with anti-low-affinity platelet factor 4 antibody. It has been determined that the supernate processes immediately after platelet aggregation induced by ionophore A23187 or thrombin contains approximately 80% low-affinity platelet factor 4, 8% beta-thromboglobulin, and 12% highly cationic immunoreactive material (platelet basic protein). Experimental evidence suggests that low-affinity platelet factor 4 is originally secreted by platelets and then converted to beta-thromboglobulin by a platelet-derived, heat-labile protease that is inhibited by phenylmethylsulfonyl fluoride.