Ar Horvath

Translocation of pp60c-src to the cytoskeleton during platelet aggregation.

The high amount of pp60c‐src in platelets has led to speculation that this kinase is responsible for tyrosine‐specific phosphorylation of cellular proteins during platelet activation by different agonists, and is, therefore, implicated in signal transduction of these cells. Unlike pp60v‐src, the association of which with the cytoskeleton appears to be a prerequisite for transformation, pp60c‐src is detergent‐soluble in fibroblasts overexpressing the c‐src gene, and its role in normal cellular function remains elusive. To gain a better understanding of the function of pp60c‐src we have investigated the subcellular distribution of pp60c‐src and its relationship to the cytoskeleton during platelet activation. Quantitative immunoblotting and immunoprecipitation have revealed that pp60c‐src is detergent‐soluble in resting platelets, while 40% of total platelet pp60c‐src becomes associated with the cytoskeletal fraction upon platelet activation. We have also shown that a small pool of pp60c‐src is associated with the membrane skeletal fraction which remains unchanged during the activation process. The interaction of pp60c‐src with cytoskeletal proteins strongly correlates with aggregation and is mediated by GPIIb/IIIa receptor‐fibrinogen binding. We suggest that the translocation of pp60c‐src to the cytoskeleton and its association with cytoskeletal proteins may regulate tyrosine phosphorylation in platelets.

4‐Thio‐deoxyuridylate‐modified thrombin aptamer and its inhibitory effect on fibrin clot formation, platelet aggregation and thrombus growth on subendothelial matrix

Summary.  Background: The consensus thrombin aptamer C15‐mer is a single‐stranded DNA of 15 nucleotides [d(GGTTGGTGTGGTTGG)] that was identified by the selection of thrombin‐binding molecules from a large combinatorial library of oligonucleotides. It is capable of inhibiting thrombin at nanomolar concentrations through binding to a specific region within thrombin exosite 1. As has been shown in our earlier studies, the 4‐thio‐deoxyuridylate (s4dU)‐containing oligonucleotides have high affinity for a number of proteins, due to the reduced hydrophilic character of the modified oligonucleotide. Methods: Three different analogs of the original thrombin‐inhibiting sequence, in which some of the thymidylate residues were replaced by 4‐thio‐deoxyuridylates, were synthesized. The inhibitory effect of modified aptamers was tested on thrombin‐catalyzed fibrin clot formation and fibrinopeptide A release from fibrinogen, thrombin‐induced platelet aggregation/secretion, and the formation of thrombus on coverslips coated with human collagen type III, thrombin‐treated fibrinogen or subendothelial matrix of human microvascular endothelial cells. Results: As compared with the C15‐mer, the analog with the sequence GG(s4dU)TGG(s4dU)G(s4dU)GGT(s4dU)GG (UC15‐mer) showed a 2‐fold increased inhibition of thrombin‐catalyzed fibrin clot formation, fibrinopeptide A release, platelet aggregation and secretion in human plasma and thrombus formation on thrombin‐treated fibrinogen surfaces under flow conditions. Concerning the inhibition of thrombin‐induced fibrin formation from purified fibrinogen and activation of washed platelets, UC15‐mer was 3‐fold and twelve‐fold more effective than C15‐mer, respectively. Conclusion: The replacement of four thymidylate residues in C15‐mer by 4‐thio‐deoxyuridylate resulted in a new thrombin aptamer with increased anticoagulant and antithrombotic properties.

Interaction between tissue transglutaminase and phospholipid vesicles

A specific interaction between purified liver transglutaminase and small unilamellar phospholipid vesicles at the lipid phase transition have been revealed. The enzyme‐induced perturbation of the bilayer is sufficient for phase transition release of encapsulated carboxyfluorescein from the vesicles. The size of the enzyme—phospholipid recombinants depends upon the protein—phospholipid ratio as shown on Sepharose 4B elution profile. The activity of transglutaminase inserted into the bilayer is greatly reduced. The interaction does not occur when the phospholipid vesicle are in the solid or liquid phase and it requires the structural integrity of the enzyme.

Tyrosine-specific phosphorylation of gpIIIa in platelet membranes

In vitro phosphorylation of platelet subcellular fractions revealed that most of the alkali‐resistant phosphoproteins and the majority of pp60c‐src were in the surface membrane fraction. An alkali‐resistant phosphoprotein of about 100 kDa was also immune precipitated by an anti‐phosphotyrosine antibody and comigrated with gpIIIa. The phosphorylation of gpIIIa, but not gpIIb, was confirmed by the comparison of reduced and non‐reduced gels, and this protein was phosphorylated exclusively on tyrosine. In contrast, both gpIIb and gpIIIa were phosphorylated when the purified complex was added to immunopurified, immobilised pp60c‐src. A synthetic peptide with partial homology to a putative tyrosine phosphorylation site in the cytoplasmic domain of gpIIIa was phosphorylated by antibody‐purified pp60c‐src. Our results indicate that tyrosine‐specific phosphorylation of gpIIIa by pp60c‐src may play a role in the regulation of platelet function.