Targeting hyperfibrinolysis: A novel prevention and reversal strategy.

Abstract Activation of the fibrinolytic cascade is a common and difficult to manage cause of massive bleeding as in trauma, but available inhibitors have shown limited benefit in severe settings. For example, the lysine analog tranexamic acid (TXA), the most widely used antifibrinolytic agent, inhibits only one step in the multi-step fibrinolytic cascade. TXA competitively inhibits the binding of plasminogen to fibrin, preventing its activation by tPA on the clot surface. However, TXA also stimulates the fibrin-independent activation of plasminogen by uPA, generating free plasmin that inactivates several coagulation factors and consumes α2-antiplasmin, predisposing to clinically relevant coagulopathy. Here we describe a novel approach to forestall the development of untoward fibrinolysis by decreasing the concentration of the fibrinolytic enzymes in the circulation of bleeding patients. This is achieved by transfusing plasminogen and tPA depleted plasma (ClearPlasma), which attenuates plasminogen activation by tPA and uPA and thereby the generation of plasmin. Reduction in plasmin generation mitigates premature cleavage of newly formed blood clots and the consumption of coagulation factors and α2-antiplasmin. To do so, plasminogen and tPA are removed from plasma using an affinity filter composed of resin bound tranexamic acid (PlasFree). A single passage of plasma over PlasFree removes over 90% of plasminogen, in addition to tPA, from normal plasma (ClearPlasma) without affecting any of the other plasma components including α2-antiplasmin. Thus, ClearPlasma is reach in coagulation factors and α2-antiplasmin and poor in fibrinolytic proteins plasminogen and tPA. To test the efficacy of ClearPlasma, studies were conducted in mice and pigs in accordance with the Institutional Animal Care and Use Committees of the Hebrew University. In the first model, anesthetized c57 black mice were bled by amputation of the tail vein. After 350 µl of blood loss, simulating severe blood loss, 300 µl of either control plasma (n = 9) or ClearPlasma (n = 11) were transfused, and bleeding was monitored for 60 minutes. Transfusing ClearPlasma decreased bleeding volume by 57 ±11% (p = 0.0194) compared with normal plasma. In the second model, 40-50 kg female domestic pigs were depleted of 700 ml of plasma by plasmapheresis to simulate moderate to severe blood loss and hypovolemia. Two hours later, a laparotomy was performed at which time a 4 x 4 cm tissue section from the right liver lobe was excised. During the laparotomy procedure, 700 ml of either control plasma (n = 6) or ClearPlasma (n = 9) were transfused and bleeding was monitored for 60 minutes. Blood loss was determined by measuring the difference in the weights of dry sponges over the ensuing 60 min. Pigs transfused with ClearPlasma show a 43 ± 14% (p = 0.0137) decrease in blood loss compared with controls. To assess potential long-term complication related to ClearPlasma, sixty min after liver laceration, the abdominal incision was closed and the animals were recovered and monitored for up to 30 days. No adverse effects related to ClearPlasma use were observed. Lastly, safety and efficacy were examined in humans comparing ClearPlasma with Fresh Frozen Plasma (FFP) in a prospective, multi-center, international, double-blind, randomized, two-arm, controlled clinical trial in patients undergoing Coronary Artery Bypass Grafting (CABG) or Valve Replacement with cardiopulmonary bypass that required transfusion of plasma. A total of 136 patients including 27 screening failures were enrolled, randomly divided in tow groups, ClearPlasma (n = 54) and FFP (n = 55) that were under follow-up for 30 days after the intervention. Patients given ClearPlasma required significantly fewer units of plasma than control group (p=0.0206) and in contrast to the controls, no significant drop in α2-antiplasmin concertation was recorded. There was a clear tendency toward reduced blood loss and prevention of massive bleeding in the ClearPlasma group, although the difference did not reach statistical significance. No device-related adverse effects were recorded throughput the 30-day follow-up period.Taken together, these studies demonstrate the feasibility, safety and efficacy of giving plasminogen and tPA-depleted plasma to prevent and reverse excess fibrinolysis and the attendant bleeding consequences