Cell therapy with platelets is a widely accepted approach for wound healing and tissue regeneration in medicine. however, with most available methods poorly concentrated platelet suspensions (up to 0.3•10 6 1/ µl) or suspensions of mostly inactivated or lost platelets are obtained. In this study, we aimed to develop a simple and effective method for preparing a suspension of native and resting platelets with over 1•10 6 1/µl. Platelet-rich plasma (PRP) was obtained from fresh blood of healthy donors (n = 5) collected using different amounts of heparin as the anticoagulant. Samples of PrP were spun down and re-suspended in auto logous blood plasma. Count and vitality of platelets in each sample were determined by aggregation study on the Solar aP2110 aggregometer. Platelet shape and cytoplasmic granularity that indicate the nativity of platelets were monitored on the CoULter ePICS XL Flow Cytometer. this study of aggregation of platelets in PrP obtained using various amounts of heparin allowed us to reduce final concentrations to the amount that effectively prevented clotting and did not affect platelet reactivi ty (5 U/ml). PRP concentrated 5 times with a total concentration of cells of 1•10 6 1/µl was able to be activated by adenosine diphosphate (aDP) (aggregation rate 54 ± 7%). the amount of cells with altered shape and granularity in concentrated suspension was not higher than 20%. This finding means that the platelets would still be able to release a number of growth factors and other biologically active compounds after stimulation or injection into tissue during cell therapy. the decrease in heparin concentrations also minimizes haemorrhage in the injection site supporting biomedical use of the suspension. A simple and effective method for preparation of highly-concentrated PRP (1.2•10 6 1/ µl) for biomedical use was developed. Aggregometry and flow cytometry proved that obtained platelets were resting and able to be activated. Being autologous, the preparation can be widely used for cell therapy without additional precautions. k e y w o r d s: cell therapy, platelets, autologous, platelet rich plasma, preparation protocol.

Earlier we reported that during the human fibrinogen to fibrin transition a neoantigenic determinant was exposed in the Bβ119-133 fragment, where a hinge locus is situated. The fibrin-specific mAb FnI-3c and its Fab-fragment with epitope in this fragment inhibited the lateral association of protofibrils. We suggested that the epitope coincided with a site involved in this process. In this work we investigated the epitope location more precisely and defined a functional role for its exposure in the hinge locus of the molecule. It was found that mAb FnI-3c bound to human, horse and rabbit fibrins, all of which have Lys in the position corresponding to human BβK130, but not to bovine and rat fibrins, which have other amino acid residues in this position, strongly suggesting that BβK130 provides the integral part of the epitope. This fact, homology data, and structural biological analysis of the amino acid sequences around BβK130 indicate that the site of interest is localized within Bβ125-135. The synthetic peptides Bβ121-138 and Bβ125-135, unlike their scrambled versions, bound to mAb FnI-3c in SPR analysis. Both peptides, but not their scrambled versions, inhibited the lateral association of protofibrils. The FnI-3c epitope is exposed after fibrinopeptide A cleavage and desA fibrin monomer formation. Structural biological analysis of the fibrinogen to fibrin transition showed a distinct increase of flexibility in the hinge locus. We propose that the structural transformation in the fibrin hinge regions leads to the conformation necessary for lateral association of protofibrils. K e y w o r d s: fibrinogen to fibrin transition, coiled-coil connector, protofibril lateral association, hinge region, neoantigenic determinant. introduction Fibrinogen is a dimer, with each subunit of the molecule being formed by three polypeptide chains: Aα, Bβ and γ. The molecule consists of a central E, two peripheral D, and two extended αC regions [1]. The central E-region consisting of (Aα1-104, Bβ1-133, γ1-72) 2 is connected to the two peripheral Dregions (Aα105-219, Bβ134-461, γ73-411) by two long flexible coiled-coil connectors (Aα48-161, Bβ79-193, γ23-135). The N-terminal parts of the connectors (Aα48-104, Bβ79-133, γ23-62) belong to the E region and the C-terminal parts (Aα105-161, Bβ134-193, γ63-135) to the D regions. A hinge locus (α99-110, β130-155, γ70-100) is located in the middle part of the coiled-coil region [2, 3]. The extended αC regions (Aα220-610) consist of unfolded flexible segments (Aα220-391) and more structured αC-domains (Aα392-610). Intermolecular binding of the fibrin polymerization sites A:a as well as putative C:c sites [4], leads to protofibril formation. The protofibrils associate laterally, initially forming fibrils and, subsequently, a three-dimensional fibrin net. At the stage of protofibrils and fibril formation, inter-protofibril binding

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