Hemolysis exacerbates hyperfibrinolysis, whereas platelolysis shuts down fibrinolysis: evolving concepts of the spectrum of fibrinolysis in response to severe injury

Abstract

Introduction: We have recently identified a spectrum of fibrinolysis in response to injury, in which there is increased mortality in patients who have either excessive fibrinolysis (hyperfibrinolysis [HF]) or impaired fibrinolysis (shutdown). The regulation of the fibrinolytic system after trauma remains poorly understood. Our group's previous proteomic and metabolomic work identified elevated red blood cell (RBC) degradation products in trauma patients manifesting HF. We therefore hypothesized that hemolysis was contributory to the pathogenesis of HF. Given the central role of platelets in the cell-based model of coagulation, we further investigated the potential role of platelet lysis in mediation of the fibrinolytic system.

Methods: Red blood cells from healthy donors were frozen in liquid nitrogen and vortexed to create mechanical membrane disruption. Platelets were prepared in a similar fashion. Assays were performed with citrated whole blood mixed ex vivo with either RBC or platelet lysates. Tissue plasminogen activator (tPA) was then added to promote fibrinolysis, mimicking the tPA release from ischemic endothelium during hemorrhagic shock. The degree of fibrinolysis was evaluated with thromboelastography. To identify the mediators of the fibrinolysis system present in RBC and platelet lysates, these lysates were passed over immobilized tPA and plasminogen affinity columns to capture protein-binding partners from RBC or platelet lysates.

Results: The addition of 75 ng/mL of tPA to whole blood increased fibrinolysis from median 30-min lysis of 1.4% (interquartile range [IQR], 0.9%-2.0%) to 8.9% (IQR, 6.5%-11.5%). Red blood cell lysate with tPA increased fibrinolysis to 20.1% (IQR, 12.5%-33.7%), which was nearly three times as much lysis as tPA alone (P < 0.001). Conversely, the addition of platelet lysate decreased tPA-mediated fibrinolysis to 0.35% (IQR, 0.2%-0.8%; P < 0.001). Affinity chromatography coupled with tandem mass spectrometry identified a number of proteins not previously associated with regulation of fibrinolysis and trauma.

Conclusion: Red blood cell lysate is a potent enhancer of fibrinolysis, whereas platelet lysate inhibits fibrinolysis. Intracellular proteins from circulating blood cells contain proteins that interact with the two key proteins of tPA-mediated fibrinolysis. Understanding the effect of tissue injury and shock on the lysis of circulating cells may provide insight to comprehending the spectrum of fibrinolysis in response to trauma.

Fig. 1. A, Hemolyzed RBCs enhance tPA-mediated fibrinolysis

WB indicates whole blood; %, percent lysate replacing whole blood. *P < 0.05 compared with tPA group. Box in upper left corner represents the Spearman ρ R² value with associated P value. A demonstrates tPA enhancement of fibrinolysis by RBC lysate. The y axis represents the median percent fibrinolysis 30 min after clots reaches their MA. At 2% replacement of whole blood with lysed RBCs, there is an increase in fibrinolysis compared with whole blood with tPA alone. This enhancement had a positive correlation on fibrinolysis with increasing the percentage of platelet lysate. Red blood cells enhanced fibrinolysis is completely reversible with tranexamic acid. B demonstrates tPA inhibition of fibrinolysis by platelet lysate. The y axis represents the median percent fibrinolysis 30 min after clots reaches their MA. At 1% replacement of whole blood with platelet lysate, RBCs decreased fibrinolysis compared with whole blood with tPA alone. This inhibition had a negative correlation on fibrinolysis with increasing the percentage of platelet lysate.

Fig. 2. A, Red blood cell lysate reduces clotting time

WB indicates whole blood; %, percent lysate replacing whole blood. *P < 0.05 compared with tPA group. Box in upper left corner represents the Spearman ρ R² value with associated P value. A demonstrates that lysate of RBCs decreases clotting time represented by the TEG parameter R. The y axis represents the mean R time in seconds. This decrease in clotting time was significant at all percentage of whole blood replaced with HL. This reduction in R time had a negative correlation with increasing the percentage of HL. This was not TXA reversible. B, Platelet lysate inhibits tPA-mediated fibrinolysis. *P < 0.05 compared with tPA group. Box in upper left corner represent the Spearman ρ R² value with associated P value. B, Platelet lysate reduces clotting time. Box in upper left corner represents the Spearman ρ R² value with associated P value. B demonstrates that lysate of platelets decreases clotting time represented by the TEG parameter R. The y axis represents the mean R time in seconds. This decrease in clotting time was significant at all percentage of whole blood replaced with PL. This reduction in R time had a negative correlation with increasing the percentage of PL.

Fig. 3. A, Red blood cell lysate increases angle in non–dose-dependent manner

WB indicates whole blood; %, percent lysate replacing whole blood. *P < 0.05 compared with tPA group. Box in upper left corner represents the Spearman ρ R² value with associated P value. A demonstrates that HL increases the rate of clot strengthening measured by the TEG parameter Angle. The y axis represents the mean angle in degrees. All percentage of HL to whole blood significantly increased the angle compared whole blood and tPA. There was not a dose effect and was not reversible by TXA. B, Platelets lysate increases angle in dose-dependent manner. B demonstrates that lysate of platelet increases the rate of clot strengthening measured by the TEG parameter angle. The y axis represents the mean angle in degrees. Platelet lysate increases the angle at all concentrations compared with whole blood and had a dose-like relationship.

Fig. 4. A, Red blood cell lysate and clot strength

WB indicates whole blood; %, percent lysate replacing whole blood. *P < 0.05 compared with tPA group. Box in upper left corner represent the Spearman ρ R² value with associated P value. A demonstrates HL relationship with clot strength measured by the TEG parameter MA. The y axis represents the mean MA. At a low dose, HL significantly increases MA compared with whole blood with tPA. Increasing the percentage of HL to whole blood had a negative correlation with clot strength. This was reversible by TXA. B, Platelet lysate increases clot strength. B demonstrates that lysate of platelet increases clot strength measured by the TEG parameter MA. The y axis represents the mean MA. Platelet lysate significantly increases clot strength compared with control at all doses. There was a positive correlation with PL dose and clot strength.

Fig. 5. Red blood cell lysate indirectly enhances tPA-mediated fibrinolysis versus platelet lysate directly shuts down tPA-mediated fibrinolysis

HL indicates RBCs from AB donor; PL, pooled platelet lysate. The figure demonstrates the changes in activity of lysate after the use of affinity chromatography to deplete proteins that interact with tPA or plasminogen. The y axis represents the change in fibrinolytic activity between whole blood mixed with tPA. All samples were run with the same final concentration of tPA (75 ng/mL). Mock (underivatized agarose beads) columns showed expected changes in fibrinolytic activity, in which lysed RBCs increased tPA-mediated fibrinolysis, whereas lysed platelets inhibited fibrinolysis. Red blood cell lysate minimally decreased in activity after running through a tPA affinity column in which proteins binding to tPA were depleted. There was a more robust decrease in activity compared with the mock column when RBC lysates were run over a column capturing proteins that bind plasminogen. Red blood cell enhancement of tPA-mediated lysis works through a mechanism that does not require a direct tPA interaction. Conversely, platelet lysates depleted of proteins that bind tPA showed a reduction in fibrinolytic activity compared with the mock column, suggesting platelet lysate directly inhibits tPA-mediated fibrinolysis.