Polymorphisms of glycoprotein Ib affect the inhibition by aurintricarboxylic acid of the von Willebrand factor dependent platelet aggregation.

Two polymorphisms of platelet glycoprotein Ib, VNTR and Thr/Met(145), regarded as the possible inherited risks factors for thromboembolic complications, have been suggested to underlie platelet response to activating stimuli. This study examined the functional significance of these polymorphisms in platelet reactivity and sensitivity to aurintricarboxylic acid (the antagonist of von Willebrand factor, vWF). To evaluate platelet function at low and high flow conditions we monitored the ristocetin-induced and vWF-mediated aggregation of isolated platelets and the platelet function analyzer collagen/ADP closure time (PFA-100 CT(CADP)), which reflects platelets' ability to adhere and aggregate in whole blood. Aurintricarboxylic acid significantly reduced ristocetin-induced platelet agglutination in a dose-dependent manner (IC(50)=3.5+/-1.9 microM). At the concentration of 100 microM it also markedly prolonged PFA-100 CT(CADP) (up to 147+/-32 s vs. 94+/-17 s in control). The efficacy of this antagonist in the inhibition of vWF-mediated platelet agglutination was approximately 1.5-fold higher in the VNTR B/Met145(+) carriers than in VNTR B/Met145(-) carriers ( P<0.05). Otherwise, no significant differences occurred between VNTR B/Met(145)-positive and B/Met(145)-negative individuals in the prolongation of closure time by ATA. These findings indicate that under certain experimental conditions VNTR-B and Met(145) alleles may contribute to the increased platelet sensitivity to some antagonists of platelet natural ligands.

Aptamer inhibits degradation of platelet proteolytically activatable receptor, PAR-1, by thrombin.

We investigated the in vitro effects of the site-directed thrombin inhibitor-a single-stranded oligonucleotide aptamer (GGTTGGTGTGGTTGG)-on thrombin proteolytic activity towards its two natural substrates: fibrinogen and platelet thrombin receptor (PAR-1). The thrombin aptamer was shown to strongly affect thrombin clotting activity at nanomolar concentrations and thrombin-dependent degradation of proteolytically activatable receptor, PAR-1, exposed on platelet surface membrane at micromolar concentrations. The incubation of PPP with thrombin in the presence of 100-1000 nM aptamer resulted in the significant concentration-dependent prolongation of thrombin time (up to fourfold, P<.0001). Aptamer significantly reduced the thrombin-induced platelet degranulation (46+/-20% inhibition at 0.15 U/ml thrombin, P<.001), as well as thrombin-mediated platelet aggregation in PRP (7+/-10% inhibition at 1 U/ml thrombin, P<.05). Furthermore, aptamer inhibited the thrombin-catalysed cleavage of PAR-1 in a dose-dependent manner, i.e., by 17%, 27% and 70%, respectively, for the concentrations of 100, 500 and 1000 nM (P<.025 by randomised block analysis; P(regression slope)<.0001). We conclude that aptamer is able to considerably attenuate thrombin proteolytic activity regardless of the molecular size of thrombin substrates. Our observations directly proved that aptamer may be successfully used for the inhibition of thrombin activity towards various physiological targets: one related to fibrin generation in the final stage of coagulation cascade, and another concerning the interaction of thrombin with its surface membrane receptor, PAR-1, in blood platelets.

Membrane lipid fluidity of blood platelets: a common denominator that underlies the opposing actions of various agents that affect platelet activation in whole blood.

Membrane lipid fluidity (MLF) is thought to play a crucial role in signal transduction and is believed to affect the responsiveness of blood platelets. In a recent study it was demonstrated that EDTA, used as the blood anticoagulant, brought about a significant increase in expression of GMP-140 antigen, and this effect was accompanied by a significant increase in platelet MLF. Moreover, this spontaneous EDTA-driven platelet activation was vastly attenuated in the presence of tissue-type plasminogen activator, which is also known to affect platelet MLF. The hypothesis was raised that the modulation of platelet membrane fluidity by EDTA might underlie platelet spontaneous activation in the presence of EDTA. To further explore the possible molecular mechanism(s) of the EDTA-dependent triggering of signal transduction pathway(s) in human blood platelets, we monitored the extent of spontaneous platelet activation in the presence of EDTA and selected platelet membrane 'fluidizers' and 'rigidizers'. A reduction in the EDTA-dependent platelet release and activation was noted, not only in the presence of rt-PA (by over 50%, P < 0.001), which acted as a rigidizer of platelet membrane fluidity (ESR h+1/h0 ratios of 5-DOXYL-Ste and 12-DOXYL-Ste decreased by 6.2%, P<< 0.0001, and 3.8%, P < 0.02, respectively), but also in the presence of other modulators of MLF, regardless of their fluidizing or rigidizing effects. Both rigidizers (procaine and lidocaine, 5-DOXYL-Ste h+1/h0 reduced by up to 6.5%, 12-DOXYL-Ste h+1/h0- by up to 4.5%, P < 0.02 or less) and fluidizers (benzyl alcohol, ethanol, 12-DOXYL-Ste h+1/h0 increased by 17.8% and 6.1%, respectively, P <<0.0001) of platelet membranes significantly depressed platelet activation (respectively, down to 1.1%, 7.7%, 6.7% and 8.5% vs control EDTA 22.9% of CD62-positive platelets). We suggest that EDTA induces alterations in membrane glycoprotein structure and affect MLF by altering lipid-protein interactions, and thus triggers signal transduction in the course of platelet activation. The resulting displacements in platelet membrane proteins, dislocation of membrane components and/or distortion of lipid-protein interactions could generate an 'outside-in' signalling that is mediated by the altered platelet MLF. Overall, it is likely that interference with the structure and conformation of selected domains of platelet membrane proteins might be the crucial mechanism by which EDTA leads to exaggerated activation of platelets in whole blood.