A genetically engineered human Kunitz protease inhibitor with increased kallikrein inhibition in an ovine model of cardiopulmonary bypass.

A recombinant human serine protease inhibitor known as Kunitz protease inhibitor (KPI) wild type has functional similarities to the bovine Kunitz inhibitor, aprotinin, and had shown a potential to reduce bleeding in an ovine model of cardiopulmonary bypass (CPB). The aim of this study was to assess KPI-185, a modification of KPI-wild type that differs from KPI-wild type in two amino acid residues and which enhances anti-kallikrein activity in a further double-blind, randomized study in an ovine model of CPB, and to compare with our previous study of KPI-wild type and aprotinin in the same ovine model. Post-operative drain losses and subjective assessment of wound 'dryness' showed no significant differences between KPI-185 and KPI-wild type, despite the significant enhancement of kallikrein inhibition using KPI-185 seen in serial kallikrein inhibition assays. These preliminary findings support the hypothesis that kallikrein inhibition is not the major mechanism by which Kunitz inhibitors such as aprotinin reduce perioperative bleeding.

Amelioration of the bleeding tendency of preoperative aspirin after aortocoronary bypass grafting.

BACKGROUND: Aspirin therapy is widely used in the treatment of cardiac disease. It has been recognized as a causative factor for increased bleeding and blood loss after open heart operations. METHODS: To determine whether high-dose aprotinin maintained its efficacy in reducing blood loss in the presence of aspirin pretreatment in patients undergoing aortocoronary bypass, we performed a double blind study on 60 adult patients. Half received high-dose aprotinin (Trasylol) and half placebo. RESULTS: Total hemoglobin loss, the primary efficacy variable was reduced from 36.1 +/- 31.4 g (mean +/- SD) to 14.1 +/- 16.0 g (p = 0.002). Blood loss was reduced intraoperatively and total loss was reduced from 837.3 mL +/- 404.9 mL to 368.7 mL +/- 164.3 mL (p < 0.001). The number of patients who did not receive any donor blood products was significantly higher in the aprotinin-treated patients (56.7% versus 23.3%, p = 0.008). Activation of the clotting cascade was significantly less in the treated patients toward the end of cardiopulmonary bypass both by measurement of thrombin-antithrombin III complex (p < 0.0001) and prothrombin fragment 1 + 2 (p < 0.0001). D-Dimer generation was significantly less from the onset of bypass and after reversal of heparin in the aprotinin-treated patients (p < 0.0001). CONCLUSIONS: High-dose aprotinin was highly effective in reducing bleeding in this high-risk group of patients. Biochemical analyses suggest the mechanism by which aspirin increases blood loss after cardiopulmonary bypass is different from the blood-preserving effects of aprotinin, which is acting as an antifibrinolytic agent.

Direct activation of factor X by monocytes occurs during cardiopulmonary bypass.

We hypothesized that monocyte procoagulant activity, which includes up-regulation of tissue factor and direct activation of factor X by CD11b. is an activator of coagulation during cardiopulmonary bypass (CPB), because recent studies have cast doubt on the presumption that the surfaces of CPB activate the intrinsic pathway. Sequential samples were taken from 17 patients undergoing cardiac surgery. During CPB a significant increase in thrombin-antithrombin complexes occurred (P < 0.0005). Factor XIIa levels increased (P < 0.005) but remained within the normal range. Total monocyte procoagulant activity was measured and a functional assay was developed to detect direct activation of factor X alone. There was a significant increase in total procoagulant activity on circulating monocytes from the start of CPB (P < 0.005) to which direct factor X activation was a major contributor (P < 0.005). Direct activation of factor X was inhibited by CD11b blocking peptides. Using flow cytometry, up-regulation of monocyte CD11b (P < 0.0005). but not up-regulation of tissue factor, was found on circulating monocytes. Monocytes adherent on the oxygenator fibres showed increased CD11b expression (P < 0.0001), but no tissue factor when assessed by fluorescent image analysis. In conclusion, direct activation of factor X through monocyte CD11b occurs during CPB and appears to contribute to thrombin generation during CPB.

Activation of coagulation and fibrinolysis during cardiothoracic operations.

BACKGROUND: During open cardiac operations using cardiopulmonary bypass, there is activation of coagulation and fibrinolysis. We assessed the separate contributions of the surgical procedure itself and cardiopulmonary bypass to this, by studying sequential samples from patients undergoing routine open cardiac operations or thoracic operations without cardiopulmonary bypass. METHODS: Activation of coagulation and the extent of fibrinolysis were measured from sequential samples obtained before the operation to 48 hours after the operation for 7 thoracic patients and 8 cardiac patients. RESULTS: In the thoracic group operation length was shorter (p = 0.002), and there was no significant increase in thrombin-antithrombin III complexes or D-dimers until 24 hours postoperatively. In contrast, there was a highly significant increase in thrombin-antithrombin III complexes (p = 0.0043) and D-dimer levels (p = 0.009) during cardiopulmonary bypass. The increase in fibrinolytic activity was caused by an increase in tissue plasminogen activator (p = 0.013). At 48 hours postoperatively, the cardiac patients had a more hypercoagulable state than thoracic patients with significantly higher levels of thrombin-antithrombin III complexes (p = 0.041) and plasminogen activator inhibitor-1 activity (p = 0.0033). CONCLUSIONS: This study suggests the major activation of coagulation and fibrinolysis seen during cardiac operations is caused by the use of cardiopulmonary bypass.

A prospective study in heart and lung transplant recipients correlating persistent Epstein-Barr virus infection with clinical events.

BACKGROUND: A 2-year prospective study was set up with 30 cardiothoracic transplant recipients to study Epstein-Barr virus (EBV) infection and immunity and their correlation with clinical events. METHODS: Regression assays were used to measure EBV-specific cytotoxic T lymphocyte (CTL) function. Tissue culture, immunoblotting, and polymerase chain reaction were used for EBV detection and isolate variation studies. RESULTS: CTL activity was significantly lower in pretransplant seropositive patients than in healthy controls (P<0.001). CTL response was undetectable in all patients during the first 6 months after transplantation, but returned at levels significantly lower than pretransplant and control levels during the second posttransplant year (P<0.001). Return of CTL function was directly correlated with time of last treated rejection episode (P<0.003) and duration of high plasma levels of cyclosporine (over 400 ng/ml; P<0.003). Significantly higher levels of EBV were detected in peripheral blood during the first 6 months than in pretransplant or control samples (P<0.05). Excretion of EBV in throat washings was significantly lower during the first 3 months when all patients were receiving acyclovir than in pretransplant and control samples (P=0.02). An increase in virus shedding was noted 3-6 months after transplantation, which was significantly higher than in pretransplant patients and controls (P<0.05). Comparison of recipients' and donors' virus isolates in 11 cases showed that seropositive recipients retained their original EBV isolate and did not acquire the donor virus. CONCLUSIONS: Immunosuppression decreased EBV-specific host immune function, which in turn favored increased EBV load in peripheral blood and increased excretion in the oropharynx. The transfer of donor virus to the seropositive recipients was not observed.

Activation of coagulation and platelets is affected by the hydrophobicity of artificial surfaces.

Clot formation is a limiting factor in the use of biomaterials. We investigated the effect of surface hydrophobicity on haemostatic activation in vitro, using five polyetherurethanes of varying surface hydrophobicity (C94, C74, C54 and C34), C94 the most, and C34, the least hydrophobic, and compared them with a commercial standard pellethane. Sterilised sacks were filled with heparinised blood, rotated at 37 degrees C for 24 h and sequential samples collected into 0.103 M sodium citrate. Thrombin generation measured by thrombin-antithrombin III complexes showed a difference between the polymers at 3 h through to 6 h (P < 0.05), C94 showing the least activation and C34 the most. Factor XIIa and D-dimer levels increased between 12 (P < 0.05) and 24 h (P < 0.01) for all polyetherurethanes. The ratio of soft:hard segments (which determine hydrophobicity) of the polyetherurethanes showed a direct relationship with the degree of activation of coagulation and fibrinolysis. There was no significant increase in monocyte tissue factor expression at 5 and 105 min. Platelet function as measured by whole blood platelet aggregation showed a reduction with pellethane and C94 after 1 h using collagen, with no changes for C34, Altering surface hydrophobicity has diverse effects on haemostatic pathways, with the most hydrophobic surfaces causing least activation of coagulation but most activation of platelets.

Transmission of donor Epstein-Barr virus (EBV) in transplanted organs causes lymphoproliferative disease in EBV-seronegative recipients.

Epstein-Barr virus (EBV) is associated with post-transplant lymphoproliferative disease (PTLD). To determine whether the donor EBV isolate is transmitted to the recipient via the allograft and causes PTLD, EBV isolates from four cases of PTLD in cadaveric heart and/or lung transplant recipients were compared with the donor isolates by PCR and DNA sequence analysis. Two recipients who were EBV seronegative at transplantation acquired an EBV isolate indistinguishable from that of the donor and developed PTLD. In contrast, in two patients who were seropositive before transplantation, the donor isolate differed from that present in PTLD of the recipient. The results suggest that the acquisition of donor EBV is a risk factor for PTLD development in a previously seronegative transplant recipient.

The effects of cardiopulmonary bypass on systemic and coronary levels of von Willebrand factor.

Von Willebrand factor antigen (vWF Ag) is a marker of endothelial injury which has been shown to rise during surgical procedures, including cardiopulmonary bypass (CPB). The aim of this study was to determine whether intermittent aortic cross-clamping during CPB causes the release of vWF Ag from the coronary vascular bed, which would suggest coronary vascular endothelial cell perturbation. Fifteen consecutive patients undergoing CPB with aortic cross-clamping during coronary artery bypass surgery and/or valve replacement by the same surgeon were studied. Paired venous and coronary sinus samples were taken pre- and post-thoracotomy, prior to cross-clamping on CPB, and 1, 5 and 10 minutes after release of the aortic cross-clamp. Plasma vWF Ag (IU/ml) was measured by ELISA. Venous vWF Ag measured prior to skin incision was 0.75 +/- 0.11 IU/ml (mean +/- SEM) and fell to 0.53 +/- 0.07 IU/ml after institution of CPB but prior to aortic cross-clamping (P < 0.01 vs pre-incision sample). Coronary sinus vWF Ag measured prior to aortic cross-clamping was 0.54 +/- 0.06 IU/ml (P = NS vs paired venous sample). At 1, 5 and 10 min after release of the aortic cross-clamp there was a progressive rise in vWF Ag in both venous and coronary sinus samples (1 min: 0.67 +/- 0.05 IU/ml vs 0.75 +/- 0.10 IU/ml, 5 min: 0.73 +/- 0.07 IU/ml vs 0.76 +/- 0.09 IU/ml, 10 min: 0.74 +/- 0.08 IU/ml vs 0.79 +/- 0.09 IU/ml; P = NS venous vs coronary sinus, respectively). Levels of vWF Ag were highest immediately prior to the termination of CPB (venous: 0.95 +/- 0.12 IU/ml; coronary sinus: 0.91 +/- 0.14 IU/ml). We conclude that cardiac surgery using CPB with aortic cross-clamping is associated with a progressive rise in coronary sinus and venous levels of vWF Ag.(ABSTRACT TRUNCATED AT 250 WORDS)