An experimental system for investigation of penetration of components of the fibrinolytic system into a plasma clot

105

109

Binding of components of the fibrinolytic system to fibrin is important for the regulation of fibrinolysis. In this study, decomposition of the fibrin network and binding of plasminogen and plasminogen activators (PAs) to fibrin during lysis of a plasma clot were investigated with confocal microscopy using fluorescein-labeled preparations of fibrinogen, plasminogen, tissue-type PA (t-PA), and two-chain urokinase-type PA (tcu-PA). Lysis induced by PAs present throughout the plasma clot was accompanied by a gradual loss of fibrin content of fibers and by accumulation of plasminogen onto the fibers. Two sequential phases could be distinguished: a phase of prelysis, during which the fibrin network remained immobile, and a phase of final lysis, during which fibers moved with a tendency to shrink and eventually disappeared. The two phases occurred simultaneously but in different locations when lysis was induced by PAs present in the plasma surrounding the clot. The zone of final lysis was located within a 5-8 microns superficial layer, where fibers were mobile, a surface-associated fibrin agglomerates appeared. Plasminogen accumulated in these agglomerates up to 30-fold as compared with its concentration in the outer plasma. t-PA was also highly concentrated in the agglomerates, and tcu-PA bound to them slightly. The zone of prelysis, where plasminogen was moderately accumulated on the immobile fibers, was located deeper in the clot. This zone was much thinner in the case of t-PA-induced lysis than in the case of tcu-PA-induced lysis, reflecting the difference in penetration of the two PAs into the clot. We conclude that under conditions of diffusional transport of fibrinolytic enzymes from outside a plasma clot, extensive lysis is spatially restricted to a zone not exceeding 5-8 microns from the clot surface. In this zone the structure of the fibrin network undergoes significant changes, and strikingly high accumulation of fibrinolytic components takes place.

Section: Methodsmentioning

confidence: 99%

Rearrangements of the Fibrin Network and Spatial Distribution of Fibrinolytic Components during Plasma Clot Lysis

Sakharov¹,

Nagelkerke

Rijken³

1996

105

109

“…Experiments with Purified Fibrin Clots-Purified fibrin clots of approximately 2.5 mm in diameter were prepared as described previously (29,30) by clotting of fibrinogen (9.2 M) with thrombin (1.4 NIH units/ml) between two parallel glass slides in a 20 mM Tris-HCl buffer, pH 7.6, containing 135 mM NaCl (TBS) and 20 mg/ml BSA (TBS-BSA). The remaining volume of the chambers (approximately 25 l) was filled with TBS-BSA and kept for 20 min at room temperature.…”

Section: Methodsmentioning

confidence: 99%

On the Mechanism of the Antifibrinolytic Activity of Plasma Carboxypeptidase B

Sakharov¹,

Plow

Rijken³

1997

205

173

The precursor of plasma carboxypeptidase B (pCPB) also known as thrombin-activable fibrinolysis inhibitor can be converted by thrombin to an active enzyme capable of eliminating C-terminal Lys-and Arg-residues from proteins. The activation is about 1000-fold more efficient in the presence of thrombomodulin (TM). We investigated the antifibrinolytic potency of maximally activated pCPB in plasma and explored the antifibrinolytic mechanism of pCPB. During clotting of plasma in the presence of 3.3 NIH units/ml thrombin and 1 g/ml soluble TM, more than 80% pro-pCPB was converted into the active form causing an increase of plasma carboxypeptidase activity from 100 units/liter (constitutive activity ascribed to plasma carboxypeptidase N) to 430 units/liter as measured with furoylacroleyl-alanyl-arginine substrate. Under these conditions, lysis of a plasma clot induced by a range of tissue-type plasminogen activator (t-PA) concentrations (0.2-2 g/ml) was retarded more than 4-fold. A considerable retardation of fibrinolysis was observed upon addition of as little as 12 ng/ml soluble TM, a concentration comparable with physiological concentrations of soluble TM in human plasma. The presence of Ca 2؉ appeared to be a critical requirement for effective activation of pro-pCPB by thrombin-TM in plasma. Plasminogen-binding sites (C-terminal lysines) on the surface of a plasmin-treated fibrin clot were eliminated within 1-3 min by plasma with maximally activated pCPB, as studied in a recently described model involving fluorescence microscopy. Confocal fluorescence microscopy showed that in the absence of TM plasminogen strongly accumulated on fibrin fibers during t-PA-induced lysis of a plasma clot. In the presence of TM (and a concomitant pro-pCPB activation), lysis was slow and was not accompanied by accumulation of plasminogen on the fibers. In conclusion, generation of active pCPB during clotting of plasma in the presence of Ca 2؉ and TM leads to a retardation of plasma clot lysis in a wide range of t-PA concentrations, from low to therapeutic, and to a fast elimination of plasminogenbinding sites on partially degraded fibrin. This is a likely mechanism for the antifibrinolytic effect of active pCPB.

“…Spatial Distribution of FITC-STA during Lysis of a Plasma Clot from the Outside-Human plasma was clotted with thrombin (final concentration, 1.4 NIH units/ml) between two parallel glass slides as described earlier (16,21). The chamber was perfused with plasma containing 0.33 M FITC-STA at 37°C.…”

Section: Methodsmentioning

confidence: 99%

“…Unlabeled Pg or STA were added in a slight molar excess (0.5 M) when indicated. After the equilibration, the clots were photographed, the negatives were scanned in a scanning densitometer (TLC Scanner CS-910, Shimadzu), and the accumulation of fluorescence inside the clots was determined using a calibration curve, as described earlier (16,21).…”

Section: Methodsmentioning

confidence: 99%

“…Binding of STA and Pg to Fibrin Clots Treated with Pl from the Outside-Purified fibrin clots of approximately 2 mm in diameter were prepared as described earlier (16,21) by clotting of Pg-free fibrinogen (final concentration, 9.2 M) with thrombin (1.4 NIH units/ml) between two parallel glass slides in a 10 mM Tris-HCl, pH 7.6, buffer containing 135 mM NaCl (TBS), and 20 mg/ml BSA (TBS-BSA). The remaining volume of the chambers (approximately 25 l) was filled with TBS-BSA and incubated for 20 min at room temperature.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Interactions between Staphylokinase, Plasmin(ogen), and Fibrin

Sakharov¹,

Lijnen

Rijken³

1996

Staphylokinase (STA), a protein of bacterial origin, induces highly fibrin-specific thrombolysis both in human plasma in vitro and in pilot clinical trials. Using fluorescence microscopy, we investigated the spatial distribution of fluorescein isothiocyanate (FITC)-labeled STA during lysis of a plasma clot and its binding to purified fibrin clots in the presence or in the absence of plasmin(ogen). STA highly accumulated in a thin superficial layer of the lysing plasma clot following the distribution of plasminogen (Pg) during lysis. Experiments with purified fibrin clots revealed that STA binds to Pg bound to partially degraded fibrin but not to Pg bound to intact fibrin. Binding of FITC-labeled STA to various forms of plasmin(ogen) in a buffer solution was studied by measuring fluorescence anisotropy. The binding constant for Glu-Pg was estimated as 7.4 M and for Lys-Pg as 0.28 M; for active-site blocked plasmin the binding constant was less than 0.05 M. The much lower affinity of STA for Glu-Pg compared with that for active siteblocked plasmin was mainly due to a lower association rate constant, as assessed by real time biospecific interaction analysis. Gel filtration of a mixture of STA with a molar excess of Glu-Pg demonstrated that STA migrated as an unbound 18-kDa protein when activation of Pg into plasmin was precluded by inhibitors of plasmin. When gel-filtered under the same conditions with plasmin, STA migrated in complex with plasmin with an apparent molecular mass of 100 kDa. Confocal fluorescence microscopy finally demonstrated that when FITClabeled STA was added to plasma before clotting, it did not bind to fibrin fibers during the first minutes (lag phase), although Pg bound to the fibers moderately. Then, both Pg and STA started to accumulate on the fibers progressively, followed by complete lysis of the clot. In conclusion, our results imply that, when STA is added to plasma, only a small percentage associates with Pg. In contrast, STA binds strongly to plasmin and to Pg, which is bound to partially degraded fibrin. These findings add a new mechanism to the known explanations for the inefficient Pg activation by STA in plasma and specify the mechanism for fibrin-dependent activation of Pg.