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ID naloge: 64 Letnik: 1999 Predmet: interna medicina
Vpliv strukture fibrinske mreže in vsebnosti trombocitov v strdku na hitrost raztapljanja s fibrinoliticnimi sredstvi
Avtor: Jožef Magdic
Mentor: doc. dr. Aleš Blinc
Z mikroskopijo na atomsko silo, ki nam daje sliko površine vzorca in nam je v danih pogojih nudila locljivost približno 5 nm, smo opazovali debelino fibrinskih vlaken in hitrost raztapljanja vlaken v fibrinskih gelih in plazemskih strdkih. Mikroskopija na atomsko silo omogoca spremljanje dinamike istega vzorca, saj ni potrebna fiksacija le-tega. Kot komplementarno metodo smo uporabili pretocne meritve, ki nam omogocajo izracun povprecne debeline vlaken in povprecne oddaljenosti med njimi, t.j. velikosti por v strdku. Zanimalo nas je, ali se debela fibrinska vlakna hitreje raztapljajo od tankih vlaken, tudi ko izkljucimo pomen transporta fibrinoliticnih encimov v strdek ter ko izkljucimo pomen hitrosti aktivacije plazminogena. Ugotoviti smo želeli, ali se fibrinska vlakna raztapljajo s precnim cepljenjem na dolocenem mestu ali s postopnim tanjšanjem celotnega vlakna. Opazovali smo, ali poteka fibrinoliza na enak nacin, ce jo sprožimo s t-PA ali s plazminom. Merili smo tudi, ali vsebnost trombocitov v strdku upocasni proces fibrinolize. Iz raztopine fibrinogena (1.8-5.5 mg/ml), plazme brez trombocitov ali plazme s trombociti (195-375 x 103/ml) smo z dodatkom trombina, na steklenem nosilcu naredili tanke strdke. Pokrili smo jih z izotonicno raztopino ali plazmo s heparinom v vodotesni celici. Po eni uri smo z dodatkom plazmina (2.5, 0.25 ali 0.17 U/ml) ali t-PA (0.2 mg/ml) sprožili fibrinolizo. Za mikroskopiranje na atomsko silo smo uporabljali Nanoscope III Atomic Force Microscope v tipalnem nacinu, s krivinskim radiem tipala 10 nm. Opazovana polja so bila velika od 0.7 x 0.7 mm do 128 x 128 mm z locljivostjo slike 512 x 512 tock. Za pretocne meritve smo strdke na enak nacin pripravili v valjastih steklenih cevkah in skoznje pretakali fiziološko raztopino pod znanim tlakom. V strdkih iz raztopine fibrinogena smo s pripravo vzorca vplivali na debelino vlaken. Iz raztopine fibrinogena v 150 mmol/l NaCl smo dobili tanka fibrinska vlakna (190 ± 25 nm) iz raztopine fibrinogena v 50 mmol/l NaCl pa debela vlakna (1,42 ± 0,19 µm). Porazdelitev debeline fibrinskih vlaken v teh vzorcih je bila unimodalna, ceprav smo v posameznem vzorcu našli vlakna razlicnih debelin. V strdkih iz plazme pa je bila porazdelitev bimodalna, saj smo v vsakem vzorcu našli mrežje izrazito debelih vlaken (v strdkih brez trombocitov 965 ± 200 nm; v strdkih s trombociti 620 ± 95 nm) in mrežje tankih vlaken (v strdkih brez trombocitov 260 ± 60 nm; v strdkih s trombociti 195 ± 30 nm). Izracun povprecne debeline vlaken iz pretocnih meritev se je bolje ujemal z meritvami iz mikroskopije na atomsko silo pri fibrinskih gelih kot pri plazemskih strdkih. Pri visoki koncentraciji plazmina (2.5 U/ml) so se tanka in debela vlakna v fibrinskih gelih raztapljala s precnim cepljenjem na dolocenem mestu, brez predhodnega tanjšanja celega vlakna, razlike v casu potrebnem za popolno precno prekinitev vlakna pa nismo zasledili (7.6 ± 3.7 min za tanka oz. 6.4 ± 4.2 min za debela vlakna). Pri nizki koncentraciji plazmina (0.17 U/ml) pa so se nekatera vlakna postopno tanjšala in sicer debela približno 3x hitreje kot tanka. Pri plazemskih strdkih brez trombocitov je fibrinoliza potekala s precnim cepljenjem vlaken, ce smo jo sprožili z dodajanjem plazmina (0.25 U/ml) ali s t-PA (0.2 mg/ml). Strdki iz plazme brez trombocitov so se ob delovanju t-PA (0.2 mg/ml) raztopili v približno 30% krajšem casu kot strdki iz plazme s trombociti (7.0 ± 3.9 min oz. 10.8 ± 2.7 min, p<0.001). Zakljucujemo lahko, da je AFM primerna metoda za opazovanje površine tankih strdkov in nam omogoca molekularno locljivost. Debela, sestavljena fibrinska vlakna se raztapljajo hitreje kot tanka vlakna. Pri visoki koncentraciji plazmina poteka raztapljanje s precno cepitvijo vlakna na dolocenem mestu, pri nizki koncentraciji tudi s postopnim tanjšanjem celotnega vlakna. Fibrinoliza poteka kvalitativno na enak nacin, ce jo sprožimo s plazminom ali s t-PA. Trombociti v strdku upocasnijo raztapljanje le-tega
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[Abstract / English version]
The effect of fibrin network structure and the presence of platelets in a clot on the rate of its lysis
Author: Jožef Magdic
Mentor: doc. dr. Aleš Blinc
We have used atomic force microscopy that measures the relief of surfaces with a resolution of about 5 nm in order to study fibrin fibre thickness and the rate of fibrinolysis in purified fibrin gels and plasma clots. Atomic force microscopy does not require fixation of samples and can therefore measure dynamic processes in real time. In addition to direct observations by atomic force microscopy we have measured flow of saline through fibrin gels and plasma clots in order to calculate the average fibrin fibre thickness and the average distance between fibres, i.e. the average pore size. We tested (a) whether thick fibrin fibres lyse at a faster rate than thinner fibres even when transport phenomena are eliminated by using very thin clots, and when the rate of plasminogen activation is eliminated by using active plasmin, (b) whether fibrin fibres lyse by transversal section at a given site, i.e., in a "cut-through" manner, or by progressive thinning of the fibre along its entire length, i.e., in a "peel-off" manner, (c) whether the pattern of fibrinolysis remains the same when fibrinolysis is initiated by plasmin in comparison to when it is initiated by tissue plasminogen activator, and (d) whether the presence of platelets in a clot slows the rate of fibrinolysis. Thin clots were prepared on glass surfaces by mixing thrombin into purified fibrinogen (1.8 - 5.5 mg/ml), platelet-depleted plasma or platelet rich-plasma (platelet count: 195 - 375 ´ 103/ml). The clots were overlaid with isotonic saline or heparinized plasma in a fluid-cell. An hour after clot formation, fibrinolysis was initiated by overlaying the clots with plasmin (2.5, 0.25 or 0.17 U/ml) or with t-PA (0.2 mg/ml). Microscopy of the samples was performed on the Nanoscope III Atomic Force Microscope operating in the tapping mode, with a cantilever tip radius of 10 nm. The field of view ranged from 0.7 x 0.7 mm to 128 x 128 mm with image resolution of 512 x 512 pixels. For permeation studies, clots were made in cylindrical glass tubes and perfused with isotonic saline under a defined pressure. In solutions of fibrinogen the concentration of NaCl had a pronounced effect on average fibrin thickness: in 150 mmol/l NaCl clots were made of thin fibrin fibres (mean 190 ± 25 nm) whereas in 50 mmol/l NaCl they were made of thick fibres (mean 1,42 ± 0,19 µm). In purified fibrin gels the distribution of composite fibre diameters was unimodal, although the range of fibre diameters was sometimes very wide. In contrast, plasma clots had a bimodal distribution of fibre diameters with networks of distinctly thick fibres (in clots from platelet-depleted plasma 965 ± 200 nm; in clots from platelet-rich plasma: 620 ± 95 nm) and networks of thin fibres (in clots from platelet-depleted plasma: 260 ± 60 nm; in clots from platelet-rich plasma: 195 ± 30 nm). The calculated average fibre thickness from permeation studies was in better agreement with AFM measurements in fibrin gels than in plasma clots. Fibrinolysis of thin and thick fibres initiated with a high concentration of plasmin (2.5 U/ml) proceeded in a "cut-through" manner with no previous thinning of the fibres. The time to complete transection did not differ between thin and thick fibrin fibres (7.6 ± 3.7 min for thin fibres vs. 6.4 ± 4.2 min for thick fibres). With a low concentration of plasmin (0.17 U/ml) some fibrin fibres became progressively thinner before complete transection occurred. The rate of fibre thinning was 3-times faster in thicker fibres than in thinner ones. Plasma clots exposed to plasmin (0.25 U/ml) and to t-PA (0.2 mg/ml) lysed predominantly by transection of the entire fibre width, i.e. in a "cut-through" manner. Clots from platelet-depleted plasma were dissolved about 30% faster than clots from platelet rich plasma after addition of 0.2 mg/ml t-PA (7.0 ± 3.9 min vs. 10.8 ± 2.7 min, p < 0.001). We conclude that AFM is a suitable method for observing surfaces of thin clots with molecular resolution. Fibrinolysis proceeds more efficiently in thick composite fibres than in thin ones. With a high concentration of plasmin fibrinolysis proceeds in a "cut-through" manner, but with a low concentration of plasmin the "peel-off" pattern is also seen. Degradation of fibrin proceeds qualitatively in the same way if it is initiated either by plasmin or by t-PA. The presence of platelets in a clot slows its rate of fibrinolysis.
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