Significant Inhibition Of Aggregation Research Articles

An antibiotic (sulfamethoxazole) stabilizes polypeptide (human serum albumin) even under extreme condition (elevated temperature)

Since aggregation of protein result into number of human diseases including diabetes mellitus, Huntington's and Alzheimer's disease, etc. Hence prevention of aggregation of a polypeptide is of great clinical importance. Human serum albumin (HSA) being major transporter serum protein was studied here in order to prevent its aggregation under extreme conditions. Sulfamethoxazole (SMZ) which is an antibiotic, caused significant inhibition of aggregation which was evident by number of biophysical techniques. Molecular docking was performed to elucidate the protein ligand binding site. In the presence of SMZ decrease in ThT, ANS and RLS fluorescence intensity suggested the inhibitory potency of this antibiotic. Further resistance to increment in the absorbance of Congo red and turbidity was observed even at elevated temperature. Circular dichroism also corroborated these results in retaining its secondary structure in the presence of SMZ. Finally the formation of aggregates, visualized under transmission electron microscopy (TEM) validated the inhibitory tendency of SMZ. Also in the parallel sets we have monitored aggregation kinetics using ThT and turbidity assay and it is noteworthy that SMZ caused maximum inhibition at protein SMZ concentration ratio of 1:30 and 1:40. Our findings would set a hallmark for designing new therapeutics for untreatable protein conformational disorders.

Targeting of the small GTPase Rap2b, but not Rap1b, to lipid rafts is promoted by palmitoylation at Cys176 and Cys177 and is required for efficient protein activation in human platelets

Rap1b and Rap2b are the only members of the Rap family of GTPases expressed in circulating human platelets. Rap1b is involved in the inside-out activation of integrins, while the role of Rap2b is still poorly understood. In this work, we investigated the localization of Rap proteins to specific microdomains of plasma membrane called lipid rafts, implicated in signal transduction. We found that Rap1b was not associated to lipid rafts in resting platelets, and did not translocate to these microdomains in stimulated cells. By contrast, about 20% of Rap2b constitutively associated to lipid rafts, and this percentage did not increase upon platelet stimulation. Rap2b interaction with lipid rafts also occurred in transfected HEK293T cell. Upon metabolic labelling with [ 3H]palmitate, incorporation of the label into Rap2b was observed. Palmitoylation of Rap2b did not occur when Cys176 or Cys177 were mutated to serine, or when the C-terminal CAAX motif was deleted. Contrary to CAAX deletion, Cys176 and Cys177 substitution did not alter the membrane localization of Rap2b, however, relocation of the mutants within lipid rafts was completely prevented. In intact platelets, disruption of Rap2b interaction with lipid rafts obtained by cholesterol depletion caused a significant inhibition of aggregation. Importantly, agonist-induced activation of Rap2b was concomitantly severely impaired. These results demonstrate that Rap2b, but not the more abundant Rap1b, is associated to lipid rafts in human platelets. This interaction is supported by palmitoylation of Rap2b, and is important for a complete agonist-induced activation of this GTPase.

Proteasome inhibitor prevents experimental arterial thrombosis in renovascular hypertensive rats.

Recent studies indicate that highly selective proteasome inhibitors can be useful in prevention of some cardiovascular events. Here we demonstrate that proteasome inhibitor, Z-Ile-Glu (Ot-Bu) Ala-Leucinal (PSI), is active in the prevention of platelet-dependent arterial thrombosis induced in renovascular hypertensive rats (two-kidney, one clip Goldblatt model, and 2K1C, n=5). The administration of PSI intravenously at a single dose of 0.3 mg/kg before induction of arterial thrombosis markedly increased carotid final flow rate, as compared to control (vehicle) group (10.36 +/- 1.8 ml/min and 1.2 +/- 1.2 ml/min, respectively), significantly decreased the wet (1.23 +/- 0.23 mg and 4.1 +/- 0.94 mg, respectively), and dry (0.46 +/- 0.145 mg and 1.46 +/- 0.39, respectively) thrombus weight, and completely prevented arterial occlusion. Moreover, platelets from PSI - treated thrombotic 2K1C rats, showed in response to collagen a significant inhibition of aggregation in the whole blood (10.26 +/- 0.6 ohms vs. 15.51 +/- 0.91 ohms in the control group). In contrast, collagen-induced platelet aggregation was not inhibited in vitro, after pre-treatment of the blood with PSI at the concentration of 10 microM that effectively inhibited the 20S proteasome activity in platelets, indicating that ex vivo anti-aggregatory effect of PSI proceeds through an indirect mechanism not associated with suppression of 20S proteasome activity in platelets. In conclusion, our in vivo findings demonstrate that proteasome inhibitor, Z-Ile-Glu(Ot-Bu)Ala-Leucinal, prevents the development of arterial thrombosis in renovascular hypertensive rats and effectively suppresses platelet aggregation by an indirect mechanism. Thus the data provide a new insight into the potential role for the proteasome-dependent pathway in cardiovascular events.

Evaluation of angiographic contrast media and platelet function with impedance aggregometry and the PFA-100TM 'platelet function analyser'

Angiographic contrast media are used in balloon angioplasty and may influence thrombotic complications of the procedure. We studied the effect of different media on platelet aggregation in whole blood using impedance aggregometry and the PFA-100 'platelet function analyser' (Dade, USA). Methods: Venous blood samples from 18 healthy volunteers were split into four aliquots and mixed with 10% normal saline control, non-ionic medium (iohexol), low-molecular weight ionic medium (ioxaglate) and high-molecular weight ionic medium (diatrizoate). Samples were studied with impedance aggregometry and the PFA-100. Results: All media caused significant inhibition of aggregation compared with control with both methods (P<0.001). Antiplatelet potency was greatest with diatrizoate, intermediate with ioxaglate and least with iohexol with both methods (P<0.01). Electron microscopy of the PFA-100 membrane demonstrated occlusion of the experimental aperture with platelet thrombus in the control. Inhibition of platelet thrombus was seen with all media, greatest with diatrizoate, intermediate with ioxaglate and least with iohexol. Conclusions: The media studied significantly inhibited platelet aggregation in vitro and potency was greater with ionic than non-ionic media. These methods use a combination of shear and chemical agonist with whole blood and may reproduce in vivo arterial conditions better than other techniques.

Coordinated inhibition of actin-induced platelet aggregation by plasma gelsolin and vitamin D-binding protein

Actin is an abundant intracellular protein that is released into the blood during tissue injury and its injection into rats causes microthrombi to form in the vasculature. This report and others have shown that actin filaments are able to aggregate platelets in an adenosine diphosphate (ADP)-dependent manner. The effects on this process of two plasma actin-binding proteins, vitamin D-binding protein (DBP) and gelsolin, were examined separately and together. The addition of DBP, a monomer-binding protein, to actin filaments did not affect their ability to induce platelet aggregation. However, severing of actin filaments with gelsolin resulted in an increased degree of platelet aggregation. Preincubation of F-actin with both gelsolin and DBP resulted in a significant inhibition of aggregation. The effects of DBP and gelsolin on actin-induced aggregation paralleled their effects on exchange of actin-bound adenine nucleotides. DBP inhibited 1, N6- ethenoadenosine 5′ triphosphate (epsilon-ATP) exchange with G-actin but not with F-actin. Gelsolin increased epsilon-ATP exchange with F-actin, which was largely abrogated by the addition of DBP. These results suggest that gelsolin's severing (and subsequent capping) of actin filaments not only results in an increase in the number of pointed filament ends but also in the dissociation of actin monomers containing ADP. Phalloidin, which stabilizes actin filaments while decreasing both monomer and nucleotide exchange, inhibited actin-induced aggregation, as well, indicating that depolymerization of actin filaments is not required to inhibit aggregation. Platelet activation by either G- or F- actin may thus be regulated by the local concentrations of the plasma actin-binding proteins gelsolin and DBP. Together, these proteins inhibit platelet aggregation in a manner that can be explained by their effects on actin's filament structure and the accessibility of its bound ADP. Depletion of DBP or gelsolin may allow actin released from injured tissues to stimulate purinergic receptors on platelets, and perhaps other cells, via its bound adenine nucleotides.

Coordinated Inhibition of Actin-Induced Platelet Aggregation by Plasma Gelsolin and Vitamin D-Binding Protein

AbstractActin is an abundant intracellular protein that is released into the blood during tissue injury and its injection into rats causes microthrombi to form in the vasculature. This report and others have shown that actin filaments are able to aggregate platelets in an adenosine diphosphate (ADP)- dependent manner. The effects on this process of two plasma actin-binding proteins, vitamin D-binding protein (DBP) and gelsolin, were examined separately and together. The addition of DBP, a monomer-binding protein, to actin filaments did not affect their ability to induce platelet aggregation. However, severing of actin filaments with gelsolin resulted in an increased degree of platelet aggregation. Preincubation of F-actin with both gelsolin and DBP resulted in a significant inhibition of aggregation. The effects of DBP and gelsolin on actin-induced aggregation paralleled their effects on exchange of actin-bound adenine nucleotides. DBP inhibited 1, N6-ethenoadenosine 5′ triphosphate (ϵ-ATP) exchange with G-actin but not with F-actin. Gelsolin increased ϵ-ATP exchange with F-actin, which was largely abrogated by the addition of DBP. These results suggest that gelsolin’s severing (and subsequent capping) of actin filaments not only results in an increase in the number of pointed filament ends but also in the dissociation of actin monomers containing ADP. Phalloidin, which stabilizes actin filaments while decreasing both monomer and nucleotide exchange, inhibited actin-induced aggregation, as well, indicating that depolymerization of actin filaments is not required to inhibit aggregation. Platelet activation by either G- or F-actin may thus be regulated by the local concentrations of the plasma actin-binding proteins gelsolin and DBP. Together, these proteins inhibit platelet aggregation in a manner that can be explained by their effects on actin’s filament structure and the accessibility of its bound ADP. Depletion of DBP or gelsolin may allow actin released from injured tissues to stimulate purinergic receptors on platelets, and perhaps other cells, via its bound adenine nucleotides.

Effects of aprotinin on anticoagulant monitoring: Implications in cardiovascular surgery

This study was designed to evaluate anticoagulant monitoring of heparin and platelet function in the presence of aprotinin. Aprotinin added to heparinized whole blood at concentrations equal to (30 μg/mL), twice, and four times that used in cardiopulmonary bypass operations synergistically elevated the activated clotting time (ACT) (536 ± 73, 651 ± 86, and 787 ± 71 seconds, respectively) over the value with heparin alone (384 ± 66 seconds) (p < 0.001). In addition, the ACT of heparin-aprotinin mixtures supplemented with protamine showed that the heparin was not completely neutralized (131 ± 12 versus 98 ± 7 seconds). Specific tests revealed that the effect on ACT caused by aprotinin is not equal to the anticoagulant effect of heparin. Thus there is a risk of underheparinization if the ACT is used as a monitor when aprotinin is present. Furthermore, protamine doses relative to the heparin concentration, and not relative to the ACT, should be used to reverse heparin. In studying the effects of aprotinin on platelet function, there was a significant inhibition of aggregation when normal platelets were supplemented with aprotinin, but not for platelets of postoperative patients. This suggests that aprotinin may interact more favorably with nonactivated platelet surfaces, reducing or inhibiting the expression of receptors. Thus it is necessary to treat a patient with aprotinin before beginning cardiopulmonary bypass. Based on these data, the effect of aprotinin on the hemostatic system and its drug interactions must be considered to optimize safety and efficacy during cardiopulmonary bypass operations.