Plasma Proteolytic Cascade Activation and Control during Neonatal Cardiopulmonary Bypass Surgery

Introduction: As blood passes through the cardiopulmonary bypass (CPB) circuit, foreign surface exposure activates cells producing tissue factor expression and microparticles that induce procoagulant and pro-inflammatory responses. In the neonate, higher pump flow rates, longer bypass times and decreased levels of endogenous plasma proteins, make plasma proteolytic cascades vulnerable to uncontrolled activation. Changes in the plasma concentrations and structures of five plasma protease inhibitors regulating coagulation, inflammation, fibrinolysis and complement were examined in neonates undergoing CPB to understand how deregulation of proteolytic cascades may contribute to excessive bleeding, which continues to be a common problem in these patients.

Methods: Blood samples were obtained at baseline (Draw 1), on CPB (Draw 2), following protamine (Draw 3) and in the intensive care unit (Draw 4). Plasma total protein and the concentrations of the serpins, antitrypsin, antithrombin, antiplasmin and C1-inhibitior, and the Kunitz inhibitor, tissue factor pathway inhibitor (TFPI) were measured. Serpin structures were examined using transverse urea gradient (TUG) gels. TFPI degradation was examined by SDS-PAGE. Excessive bleeding was defined by meeting one or more of the following: ≥ 84 mL/kg total chest tube output (CTO) in the first 24 hours, ≥ 7 mL/kg/hr CTO for ≥2 consecutive hours in the first 12 hours, or re-exploration in the first 24 hours for bleeding or tamponade.

Results: Of 44 neonates, 16 (36%) were classified as bleeders. The total plasma protein concentration was 60% that of adults at Draw 1. Adult plasma transfusion increased total protein by 43%, 59% and 32% at Draws 2, 3 and 4. The concentration of each inhibitor changed over the course of surgery (p<0.001 for all). Antitrypsin, an inhibitor of inflammation, and C1-inhibitor, an inhibitor of complement, are acute phase proteins. C1-Inhibitor increased in neonates by 56% and 81% at Draws 2 and 3, returning to baseline at Draw 4. However, antitrypsin decreased 30% between Draws 1 and 2, and remained low at Draws 3 and 4, suggesting it is consumed during surgery. Antithrombin and antiplasmin inhibit coagulation and fibrinolysis, respectively. Changes in antithrombin concentration mirrored those of total protein, while antiplasmin remained stable or decreased, again suggesting consumption. TFPI inhibits coagulation. It is a heparin-releasable protein that increased 5-fold at Draw 2, and remained 4-fold and 3-fold elevated following protamine at Draws 3 and 4. Comparisons of neonates who bled with those who did not revealed that antithrombin was higher in those bleeding at Draw 2 (p=0.031). There was no difference between groups in other inhibitors. TUG gels produce a unique unfolding signature for each serpin and differentiate virgin, proteolytically cleaved, protease complexed and polymerized forms. TUG signatures revealed only virgin antitrypsin, C1-inhibitor and antiplasmin at all 4 draws. Heparin administration at Draw 2 produced antithrombin-FXa and antithrombin-thrombin complexes, as well as proteolytically cleaved forms of antithrombin. These were also variably present at Draws 3 and 4. Western blots for TFPI revealed the appearance of degraded forms at Draws 3 and 4 in some patients.

Conclusions: C1-inhibitor concentration increased during surgery, consistent with it being an acute phase reactant. Changes in antithrombin concentration mirrored the conversion to adult plasma, while antitrypsin and antiplasmin decreased in concentration suggesting consumption and clearance. Structural analysis of serpins, other than antithrombin, revealed only the virgin form, indicating the absence of widespread, unregulated proteolysis by plasma proteolytic cascades. Antithrombin displayed complexed and cleaved forms following heparin administration, consistent with its role as an anticoagulant during surgery. TFPI increased 5-fold following heparin administration and remained 3-fold elevated at Draw 4. A portion of this elevation could be attributed to proteolytic degradation, but it is unclear why levels remained so high following protamine reversal. TFPI levels were not different between neonates who bled or not. In contrast, elevated antithrombin levels at Draw 2 were more common in neonates who bled, suggesting that administration of recombinant antithrombin may worsen bleeding in these patients.