The spatial dynamics of fibrin clot dissolution catalyzed by erythrocyte-bound vs. free fibrinolytics

Abstract

Summary background: Coupling fibrinolytic plasminogen activators to red blood cells (RBCs) has been proposed as an effective, yet safe method of thromboprophylaxis, because of increased circulation lifetime and reduced propensity to induce hemorrhage by selectivity for nascent thrombi rather than pre-formed hemostatic clots.

Objectives and methods: We used confocal microscopy of fluorescently labeled fibrin and erythrocytes in plasma-derived clots to study the spatial dynamics of lysis catalyzed by RBC-coupled vs. free plasminogen activators (RBC-PA vs. PA).

Results: Clot lysis catalyzed by free PA progressed gradually and uniformly. In contrast, distinct holes formed surrounding RBC-PA while the rest of the clot remained intact until these holes enlarged sufficiently to merge, causing sudden clot dissolution. Compared with naïve RBCs within clots lysed by free PA, RBC-PA moved faster inside the fibrin network prior to clot dissolution, providing a potential mechanism for spatial propagation of RBC-PA induced lysis. We also showed the focal nature of fibrinolysis by RBC-PA as dense loading of PA onto RBCs initiates more efficient lysis than equal amounts of PA spread sparsely over more RBCs. In an in vitro model of clots exposed to buffer flow, incorporated RBC-PA increased permeability and formed channels eventually triggering clot dissolution, whereas clots containing free PA remained intact.

Conclusions: Clot lysis by RBC-PA begins focally, has a longer lag phase when measured by residual mass than homogeneous lysis by PA, is propagated by RBC-PA motility and provides more effective clot reperfusion than free PA, making RBC-PA attractive for short-term thromboprophylaxis.

Fig. 1

Schematic diagram of the plasminogen activator (PA) fusion protein's interaction with glycophorin A on the surface of Mouse red blood cells (mRBCs).

Fig. 2

Lysis by red blood cell-plasminogen activator (RBC-PA) differs from lysis by free PA. Lysis by free PA (0.00820 μM, final conc) + 1% human RBCs (hRBCs) (A) or 1% RBC-PA (B) as a function of time visualized by spinning disk confocal microscopy. Times are % of the total time required to achieve complete clot dissolution (25 min for free PA and 41 min for RBC-PA). These images are representative of six RBC-PA and nine free PA experiments.

Fig. 3

Lysis by red blood cell-plasminogen activator (RBC-PA) differs quantitatively from lysis by free PA. Lysis catalyzed by free PA (0.00902 μM, final conc) + 2% hRBCs (A) or 2% RBC-PA (B) visualized by confocal microscopy. In these particular images, representative of 10 free PA and six RBC-PA experiments, both networks achieved dissolution at 31 min. (C) A plot of average % clot lysis vs. a relative scale of % time to complete network dissolution. Error bars are standard error of the mean. *P < 0.05 for comparison of RBC-PA with free PA (independent t-test; n = 6 for RBC-PA and n =10 for free PA).

Fig. 4

Red blood cell-plasminogen activator (RBC-PA) are more mobile within the clot prior to its destruction than are naïve RBCs with free PA. RBC velocity plotted vs. % time requiredtoachieve complete clot dissolution. Error bars are standard error of the mean. *P < 0.05 for comparison of RBC-PA movement with RBCs + free PA movement at the same point in time (independent t-test; n =63 RBCs from seven experiments for RBC-PA; n = 102 RBCs from 10 experiments for free PA). Brackets are used to show intervals in which P < 0.05 for comparison of RBC-PA with free PA at every time point within the indicated region.

Fig. 5

Densely-loaded red blood cell-plasminogen activator (RBC-PA) triggers more profound fibrinolysis than a similar dose of sparsely-loaded RBC-PA. Data are shown as % clot lysis vs. time (min). Fibrinolysis was monitored after addition of three different total amounts of PA loaded either sparsely or densely onto RBC-PA. Results are representative of four individual experiments.

Fig. 6

Red blood cell-plasminogen activator (RBC-PA), but not free PA, incorporated into clots provides reperfusion in a flow system. (A) Fibrinolysis monitored by quantitative confocal microscopy triggered by free PA (0.0344 μM, final conc) + 2% hRBCs, 2% RBC-PA (equivalent to 0.136 μM uPA), or non-lysing control + 2% hRBCs in a static model system. (B) Permeability constant for liquid perfusion in the flow system for the same samples as (A). (C) Permeability for cellular components, represented as the number per μL of buffer-derived RBCs that permeated the clot during lysis. Plots are representative of six (A, B) and three (C) independent experiments.