Addition of the MSA1 signal and anchor sequences to the malaria merozoite surface antigen 1 C-terminal region enhances immunogenicity when expressed by recombinant vaccinia virus.

Genes encoding four different C-terminal fragments of a Plasmodium falciparum merozoite surface antigen were generated: MSA1C-(Si,A), containing signal and anchor regions of MSA1; MSA1C-(Si,nA), containing the signal but not the anchor; MSA1C-(nSi,A), containing the anchor but not the signal, and MSA1C-(nSi,nA) containing neither the signal nor the anchor region. Each gene was inserted into the thymidine kinase region of vaccinia virus, under the control of a synthetic strong early/ late promoter. When the plasmodial genes were expressed in cells infected by the recombinant vaccinia virus, the two proteins containing the signal region were transported to the surface of infected cells. Infection of mice and rabbits with the latter recombinant viruses stimulated C-terminal-specific antibody levels that were 10-80-fold higher than those induced by the two recombinant viruses without the signal region. The combination of the signal and anchor regions with the C-terminal MSA1 protein also generated the most effective neutralization in a P. falciparum invasion assay.

Inhibition of platelet aggregation in whole blood by alcohol.

Our previous studies have demonstrated that addition of moderate volumes of absolute alcohol (34-170 mM final concentration) to whole blood produces concentration-dependent platelet aggregation, due to release of adenosine diphosphate (ADP) from erythrocytes. We have now investigated the effects of exposure of blood to ethanol by a more "physiologic" protocol, in which 7.8% (w/v) alcohol is added to achieve a final concentration of 1 to 85 mM in human and rat blood or platelet rich plasma (PRP). The effects of short incubation with alcohol on platelet aggregation induced by ADP, collagen and arachidonic acid were examined by the impedance method of aggregometry. Aggregation induced by collagen in PRP of either species was significantly inhibited by 85 mM ethanol, with concentrations as low as 4.25 mM inhibiting the response to collagen in rat whole blood. ADP stimulated only primary, reversible aggregation in rat PRP and whole blood, and these responses were unaffected by alcohol. Human platelets responded to ADP with irreversible aggregation, which was significantly attenuated by 85 mM ethanol in whole blood but not PRP. Arachidonic acid evoked irreversible platelet aggregation in all four preparations; this was significantly inhibited by the high dose ethanol in human and rat PRP, but not whole blood. In contrast to our earlier studies with absolute ethanol, there was no evidence of hemolysis (and therefore, ADP release from red blood cells) using the current protocol. The results of these experiments show that alcohol, at physiologically relevant concentrations, has an inhibitory effect on secondary platelet aggregation responses to some agonists in whole blood as well as PRP, possibly by its previously demonstrated effects on arachidonic acid release by phospholipases. The possibility remains to be considered that other blood cells might contribute to the effects of alcohol on platelet aggregation in whole blood.

Immunoreactive neuropeptide Y (NPY) in plasma and platelets of rat and mouse strains and human volunteers.

Immunoreactive-neuropeptide Y (i-NPY) is present in platelets of rats, and has recently been demonstrated to be authentic rat NPY based on its amino acid sequence. This potent vasoconstrictor and putative smooth muscle mitogen is released during platelet activation, suggesting a role in platelet-vascular interactions. We have now extended this work to several strains of rats and mice, and humans of both sexes. Among mice, strains in which NPY mRNA has been demonstrated in megakaryocytes have markedly higher levels of i-NPY (0.63-1.11 pmol/ml in NZB/B1NJ, NZBWF1/J, BXSB/MpJYaa, BALB/cJ) in platelet rich plasma (PRP) than other strains (DBA/2J, CBA/J, C3H/HeJ, MRL/MpJ-lpr, C57BL/6J; each < 0.02 pmol/ml). In rats, high content of i-NPY was observed in PRP and platelets of all strains examined (Sprague-Dawley, Wistar, Wistar Kyoto). i-NPY level was 30.6, 3.7 and 10.1 pmol/ml in PRP of the three strains, respectively. In humans, low levels of i-NPY occur in plasma and platelet fractions compared to rodents (0.069 and 0.048 pmol/ml in male and female PRP, respectively), but they, too, have greater i-NPY in platelet rich plasma and platelets than in platelet poor plasma. Assuming this is authentic NPY, platelet-derived NPY might have a role in pathophysiological states involving activation of platelets in humans.

Comparison of verapamil and nifedipine in thrombosis models.

Calcium blockers and calmodulin antagonists have been reported to inhibit the aggregation of blood platelets in vitro. In the present study, the effects of two calcium blockers, verapamil and nifedipine, were compared in several rodent thrombosis models. In rat and mouse platelet-rich plasma, preincubation with either verapamil or nifedipine had a dose-dependent inhibitory effect on collagen-induced aggregation (P less than 0.01). The concentration required for 50% inhibition of rat platelet aggregation was 0.91 X 10(-4) M for verapamil and 1.77 X 10(-4) M for nifedipine. In in vivo thrombosis models in mice, acute pretreatment with nifedipine had a significant, dose-dependent protective effect (P less than 0.05). At a dose of 500 micrograms/kg, nifedipine inhibited thrombotic sudden death provoked by arachidonic acid, a thromboxane agonist (U46619), or a combination of collagen and epinephrine. In vivo platelet depletion induced by U46619 was also inhibited by this calcium blocker. Thus, nifedipine is protective against a variety of thrombotic stimuli, and its antiplatelet aggregatory effect apparently extends to the in vivo situation. In contrast, no in vivo antithrombotic activity was observed for verapamil. Two additional calcium blockers, perhexilene and diltiazem, and three calmodulin antagonists, W-7, chlorpromazine, and trifluoperazine, were also tested in the U46619-induced thrombotic sudden death model. Of these, only diltiazem (5 and 10 mg/kg) had an acute protective effect.

Sex differences in mouse platelet aggregation.

The role of platelets in the sex difference observed in mouse thrombosis models was evaluated by examining platelet diminution in vivo after thrombotic challenge, and aggregation of mouse platelets in PRP. A fall in platelet count was observed in both sexes after i.v. injection of either arachidonic acid or the thromboxane agonist, U46619. Platelet diminution induced by high dose arachidonate (50 mg/kg) was significantly greater in males compared to female mice. Responses to U46619 were similar in both sexes. In PRP, male platelets exhibited a greater response than female platelets to both ADP (15 uM) and arachidonate (0.3 mM), but not to U46619 (4.6 and 6.9 uM). These results suggest that the gender difference in arachidonate-induced sudden death, in which males are more susceptible than females, is related to a sex difference in mouse platelet function.