Bogdan Walkowiak

Comparison of platelet aggregability and P-selectin surface expression on platelets isolated by different methods.

Three methods commonly used for isolation of blood platelets from plasma were compared. Platelets were isolated by: 1) a washing method; 2) a method of metrizamide-gradient centrifugation; 3) a modified method of gel-filtration. The last method employed BSA-Sepharose gel instead of routinely used Sepharose gel saturated with BSA. BSA-Sepharose gel was prepared by covalent binding of thermally deactivated BSA to CNBr-activated Sepharose 2B. In contrast to platelets isolated by the other methods, an aggregability of the gel-filtered platelets and control platelets in plasma, both activated with ADP, were comparable. When expression of P-selectin on the surface of freshly isolated platelets was examined, the gel-filtered platelets exhibited the same extent of fluorescence signal as platelets in the citrated blood, whereas platelets isolated by the other methods exhibited twice the extent of the signal. The methods involving the centrifugation process cause a low but a significant platelet activation.

MICROPLATE READER - A CONVENIENT TOOL IN STUDIES OF BLOOD COAGULATION

Results of platelet aggregation measured with a dual channel aggregometer and with a microplate reader are compared. Platelets in plasma were activated by ADP and by collagen, and thrombin was used for the aggregation study of gel filtered platelets. The results obtained with both instruments were quantitatively and qualitatively similar. A microplate reader allowed a simultaneous measurement of a high number of samples with a high degree of reproducibility. The same instrument can be useful in other coagulology studies. Results of citrated plasma clotting by thrombin or by recalcination together with results of platelet counting, both obtained with a microplate reader, are presented in this report as well.

Platelet membrane lipid fluidity and intraplatelet calcium mobilization in type 2 diabetes mellitus.

Abstract: The aim of the present study was to relate the impairments in calcium mobilization and/or release to the altered membrane dynamics in platelets from patients with type 2 diabetes mellitus. Higher expression of P‐selectin (1.4‐fold, NS) and the reduction in GPIbα expression (by 27.8 ± 16.7%, p < 0.0002), as well as the increased fractions of platelet microparticles (p < 0.03), reflected more intensified platelet release reaction in diabetic platelets. Overall, diabetic platelets appeared more vulnerable to stimuli facilitating calcium mobilization (by 41%, p < 0.01) and less susceptible to preventive effects of the agents hampering calcium release from intraplatelet storage pools (by 38%, p < 0.01). Both the increased calcium mobilization from intraplatelet storage pools and higher levels of intracellular free calcium in the presence of procaine in diabetic platelets correlated with the reduced platelet membrane lipid fluidity (resp. pR < 0.03 and pR < 0.015). We conclude that the biophysical state of platelet membrane components in diabetes mellitus is the crucial determinant of platelet hyperfunction and probably contributes to the intensified calcium mobilization in diabetic platelets. The depressed preventive effects of procaine on platelet release reaction and calcium mobilization in diabetic platelets may result from the primary dislocations and/or distortions of membrane components caused by the diabetic state.

Technical concept of patient-specific, ultrahigh molecular weight polyethylene orbital wall implant

INTRODUCTION The authors have been using patient-specific implants since 2006 and are constantly looking for new reconstructive materials, in order to create precise implants for orbital reconstruction. Such materials should be biocompatible and stable in the human body, as well as easy to machine and form into complex 3D shapes. Biocompatible ultrahigh molecular weight polyethylene (UHMW-PE) has several unique properties including high impact strength and a low friction coefficient that result in self-lubricating and thus non-sticking surfaces after processing. AIM To present the concept of a patient-specific, UHMW-PE orbital wall implant. MATERIALS AND METHODS The material used to manufacture the orbital implant was UHMW-PE converted into a solid block of medical polymer from a powder material. A delayed treatment unilateral orbital fracture case was chosen for reconstruction with patient-specific orbital wall implant. On the basis of computerized tomography, a virtual model of both orbits was prepared. The injured orbit was significantly enlarged due to dislocation of its walls. The 3D model of the facial skeleton was symmetrically divided into two parts. This resulted in two models - left and right orbit, then the uninjured orbit was superimposed onto the contralateral side. As a result two surfaces were created; the outer surface (taken from the injured orbit) was used to design the outer surface of the implant, and the inner (taken from the uninjured orbit) for the inner surface. By combining both these surfaces it was possible to determine the unique shape and thickness of the UHMW-PE implant that would allow for accurate reconstruction of the orbit. Following this, the CAD model was transferred to CAM software and a numerical code for a 5-axis milling machine was generated. The manufactured implant was sterilized in gas plasma and used to reconstruct three orbital walls. RESULTS The thickness of the manufactured implant ranged from 0.2 mm to 1.5 mm and was successfully inserted via transconjunctival approach. The lower, medial and lateral walls were reconstructed. The correct position of the right eyeball was re-established by moving it upward and medially, which resulted in enophthalmos and diplopia correction. The described method features several advantages: accurate reconstruction of the original shape of the orbit, precise modification of local implant thickness during design of the CAD model, structural globe support combined with a thin implant, the possibility of repairing large orbital floor defects, corrections using scissor/scalpel during surgery are relatively uncomplicated, low level of morbidity, smooth edges and gradual, controlled variations in implant thickness between different regions. Disadvantages: changes to the curvature of the implant cannot be made during surgery, implant may require fixing with screws to be stabilized during the early phase of healing, long time required to design and manufacture implants (pre-op) and also UHMW-PE implants are radiolucent and cannot be imaged using X-rays. CONCLUSION UHMW-PE appears to have numerous advantages as a material for precise reconstruction of the orbits. Such patient-specific implants are durable, can even be used to reconstruct very thin walls, do not exhibit the high degree of morbidity typical for autogenous bone grafts and result in restoration of vision function.

Release of calcium and P-selectin from intraplatelet granules is hampered by procaine

The inhibition of platelets by some local anaesthetics has been related to the modulation of platelet membrane lipid fluidity, and one of these compounds, procaine, has been proven to be particularly effective inhibitor. In the present study, we examined the effect of procaine on the mobilization of intracellular granule contents in isolated washed platelets. We revealed that the presence of 10 mg/ml procaine significantly hampered platelet release reaction, as demonstrated by the significant reduction in the expression of platelet P-selectin (CD62) on one hand, and significantly enhanced expression of GPIb alpha (CD42b) antigen on the other, following either 1 hour incubation of washed platelets at room temperature (%CD62: 37.1+/-6.8% of control incubated without procaine, p<<0.0001; %CD42b: 116.2+/-6.3% of control, p<0.0001) or activation of whole blood platelets with ADP, TRAP, or thrombin. Procaine, which acted as a rigidizer, significantly decreased platelet membrane fluidity (ESR h(+1)/h0 ratio of 5-DOXYL-Ste reduced down to 93.1+/-3.7% of control, p<0.001). In washed Fura-2-loaded platelets procaine not only brought about the significantly reduced Ca2+ release from intraplatelet storage pools after platelet stimulation with 15 micromol/l ADP (25.3+/-12.5% of control, p<0.001), but also it significantly increased the reduction in Ca2+ concentration upon the addition of Ca2+ chelator, EDTAK2 (48.9+/-13.5% vs. 40.9+/-12.1% of initial [Ca2+]i concentration, p(1,alpha)<0.025). Overall, procaine considerably reduced calcium mobilization from intraplatelet storage pools and Ca2+ efflux across platelet membrane. Based on these data, we suggest that the preventive effects of procaine on platelet release reaction and calcium mobilization might relate to the changes in the organization of membrane components embedded into a lipid bilayer, which are crucial in triggering of platelet release reaction. Procaine-mediated dislocations of some membrane components and/or distortion of lipid-protein interactions could generate a steric hindrance, which might interfere with platelet signal transduction, thus leading to impaired mobilization of Ca2+ and other components from intraplatelet storage pools.

Adhesion, activation, and aggregation of blood platelets and biofilm formation on the surfaces of titanium alloys Ti6Al4V and Ti6Al7Nb†

Titanium alloys are still on the top list of fundamental materials intended for dental, orthopedics, neurological, and cardiovascular implantations. Recently, a special attention has been paid to vanadium-free titanium alloy, Ti6Al7Nb, that seems to represent higher biocompatibility than traditional Ti6Al4V alloy. Surprisingly, these data are not thoroughly elaborated in the literature; particularly there is a lack of comparative experiments conducted simultaneously and at the same conditions. Our study fills these shortcomings in the field of blood contact and microbiological colonization. To observe platelets adhesion and biofilm formation on the surfaces of compared titanium alloys, fluorescence microscope Olympus GX71 and scanning electron microscope HITACHI S-3000N were used. Additionally, flow cytometry analysis of platelets aggregation and activation in the whole blood after contact with sample surface, as an essential tool for biomaterial thrombocompatibility assessment, was proposed. As a result of our study it was demonstrated that polished surfaces of Ti6Al7Nb and Ti6Al4V alloys after contact with whole citrated blood and E. coli bacterial cells exhibit a considerable difference. Overall, it was established that Ti6Al4V has distinct tendency to higher thrombogenicity, more excessive bacterial biofilm formation and notable cytotoxic properties in comparison to Ti6Al7Nb. However, we suggest these studies should be extended for other types of cells and biological objects.

The blood platelet proteome is changed in UREMIC patients

Comparative analysis of the protein profile of blood platelets isolated from nondialyzed and hemodialyzed uremic patients and healthy controls has been performed. These preliminary results indicate markedly changed expression of several proteins in blood platelets from both groups of patients compared with controls, with dramatic changes in hemodialyzed patients in the over-expression of low molecular peptides with a very wide range of pI values.

Platelet fibrinogen receptors in migraine patients.

SYNOPSIS

Concentration of RGDS-containing degradation products in uremic plasma is correlated with progression in renal failure

A concentration of protein degradation products containing the RGDS sequence, which could contribute to a lower reactivity of uremic platelets, has been estimated in both uremic (n = 16) and control (n = 7) plasmas. Degradation products and other small molecules were separated from plasma by filtration through AMICON YM-10 filter. RGDS antigen was determined in filtered material using the radioimmunoassay method based on monospecific anti-RGDS rabbit polyclonal antibodies. The concentration of RGDS-containing degradation products in uremic plasma ranged from 0.8 to 353 nM with mean value 58.6 +/- 24.9 nM and was higher than in control (0.7 to 5.9 nM, mean value 2.1 +/- 0.9 nM). Moreover, the level of RGDS-antigen positively correlated with plasma creatinine concentration (R = 0.87, p < 0.001). The filtered material showed an inhibitory effect on fibrinogen binding to control platelets in respect to RGDS-antigen concentration. We conclude that the elevated concentration of RGDS-containing degradation products in uremic plasma is partially responsible for bleeding tendency in renal failure.

Inhibition of ADP-triggered blood platelet aggregation by diadenosine polyphosphate analogues.

The synthesis and biological evaluation of new diadenosine polyphosphate analogues on blood platelet aggregation are reported. The most active are compounds with a sulfur atom replacing one or both non-bridging oxygens at phosphorus bound to adenosyl residues and hydroxymethyl groups of bis(hydroxymethyl)phosphinic acid.