Acylated 1H-1,2,4-Triazol-5-amines Targeting Human Coagulation Factor XIIa and Thrombin: Conventional and Microscale Synthesis, Anticoagulant Properties, and Mechanism of Action.

We herein report the conventional and microscale parallel synthesis of selective inhibitors of human blood coagulation factor XIIa and thrombin exhibiting a 1,2,4-triazol-5-amine scaffold. Structural variations of this scaffold allowed identifying derivative 21i, a potent 29 nM inhibitor of FXIIa, with improved selectivity over other tested serine proteases and also finding compound 21m with 27 nM inhibitory activity toward thrombin. For the first time, acylated 1,2,4-triazol-5-amines were proved to have anticoagulant properties and the ability to affect thrombin- and cancer-cell-induced platelet aggregation. Performed mass spectrometric analysis and molecular modeling allowed us to discover previously unknown interactions between the synthesized inhibitors and the active site of FXIIa, which uncovered the mechanistic details of FXIIa inhibition. Synthesized compounds represent a promising starting point for the development of novel antithrombotic drugs or chemical tools for studying the role of FXIIa and thrombin in physiological and pathological processes.

Anticoagulation With an Inhibitor of Factors XIa and XIIa During Cardiopulmonary Bypass.

BACKGROUND: Exposure of blood to polyanionic artificial surfaces, for example, during cardiopulmonary bypass (CPB), induces a highly procoagulant condition requiring strong anticoagulation. Unfractionated heparin (UFH) is currently used during CPB but can lead to serious bleeding complications or development of a hypercoagulable state culminating in life-threatening thrombosis, highlighting the need for safer antithrombotics. Ixodes ricinus contact phase inhibitor (Ir-CPI) is a protein expressed by I. ricinus ticks, which specifically inhibits both factors XIIa and XIa, 2 factors contributing to thrombotic disease while playing a limited role in hemostasis. OBJECTIVES: This study assessed the antithrombotic activity of Ir-CPI in animal contact phase-initiated thrombosis models, including CPB. The safety of Ir-CPI also was evaluated. METHODS: The authors evaluated the antithrombotic activity of Ir-CPI by using in vitro catheter-induced clotting assays and rabbit experimental models of catheter occlusion and arteriovenous shunt. During CPB with cardiac surgery in sheep, the clinical applicability of Ir-CPI was investigated and its efficacy compared to that of UFH using an uncoated system suitable for adult therapy. Taking advantage of the similar hemostatic properties of pigs and humans, the authors performed pig liver bleeding assays to evaluate the safety of Ir-CPI. RESULTS: Ir-CPI prevented clotting in catheter and arteriovenous shunt rabbit models. During CPB, Ir-CPI was as efficient as UFH in preventing clot formation within the extracorporeal circuit and maintained physiological parameters during and post-surgery. Unlike UFH, Ir-CPI did not promote bleeding. CONCLUSIONS: Preclinical animal models used in this study showed that Ir-CPI is an effective and safe antithrombotic agent that provides a clinically relevant approach to thrombosis prevention in bypass systems, including highly thrombogenic CPB.

An anticoagulant peptide from Porphyra yezoensis inhibits the activity of factor XIIa: In vitro and in silico analysis.

Thrombosis represents a major cause of morbidity and mortality around the world. Peptides isolated from natural sources have been proven to have anticoagulant and antithrombotic properties. VITPOR AI, a 16-mer peptide, isolated from Porphyra yezoensis was reported to have anticoagulant property. In this study, the coagulation factor XIIa activity in the presence of VITPOR AI was determined. Molecular modelling was performed to investigate the interaction between peptide and FXIIa. The structure of the peptide was predicted using PEP-FOLD3 server and simulated by molecular dynamics (MD) using GROMACS package. Molecular docking was carried out using peptide-protein docking software, pepATTRACT and its stability was confirmed by MD simulations. The chromogenic substrate assay revealed that the peptide inhibited the amidolytic activity of FXIIa with IC50 of 70.24 µM. The docking result showed peptide interactions through hydrogen bonds with Pro 96, Tyr 99, Glu 146, Gly 193 and Ser 195 of FXIIa. The MD simulation demonstrated that the peptide's binding with the FXIIa was stable as it did not move away from its binding region throughout the simulation period of 100 ns Moreover, MM/PBSA analysis also indicated a stable binding between the protein and peptide. These results suggest that the inhibition of the FXIIa activity might be due to binding of the peptide to oxyanion hole of the catalytic site. Thus, VITPOR AI could be explored as a potent anticoagulant which inhibits only intrinsic pathway of coagulation cascade but does not affect the extrinsic pathway.

A One-Pot "Triple-C" Multicyclization Methodology for the Synthesis of Highly Constrained Isomerically Pure Tetracyclic Peptides.

A broadly applicable one-pot methodology for the facile transformation of linear peptides into tetracyclic peptides through a chemoenzymatic peptide synthesis/chemical ligation of peptides onto scaffolds/copper(I)-catalyzed reaction (CEPS/CLIPS/CuAAC; "triple-C") locking methodology is reported. Linear peptides with varying lengths (≥14 amino acids), comprising two cysteines and two azidohomoalanines (Aha), were efficiently cyclized head-to-tail by using the peptiligase variant omniligase-1 (CEPS). Subsequent ligation-cyclization with tetravalent (T41/2 ) scaffolds containing two bromomethyl groups (CLIPS) and two alkyne functionalities (CuAAC) yielded isomerically pure tetracyclic peptides. Sixteen different functional tetracycles, derived from bicyclic inhibitors against urokinase plasminogen activator (uPA) and coagulation factor XIIa (FXIIa), were successfully synthesized and their bioactivities evaluated. Two of these (FF-T41/2 ) exhibited increased inhibitory activity against FXIIa, compared with a bicyclic control peptide. The corresponding hetero-bifunctional variants (UF/FU-T41/2 ), with a single copy of each inhibitory sequence, exhibited micromolar activities against both uPA and FXIIa; thus illustrating the potential of the "bifunctional tetracyclic peptide" inhibitor concept.

Factor XII as a Therapeutic Target in Thromboembolic and Inflammatory Diseases.

Coagulation factor XII (FXII, Hageman factor) is a plasma protease that in its active form (FXIIa) initiates the procoagulant and proinflammatory contact system. This name arises from FXII's unique mechanism of activation that is induced by binding (contact) to negatively charged surfaces. Various substances have the capacity to trigger FXII contact-activation in vivo including mast cell-derived heparin, misfolded protein aggregates, collagen, nucleic acids, and polyphosphate. FXII deficiency is not associated with bleeding, and for decades, the factor was considered to be dispensable for coagulation in vivo. However, despite the fact that humans and animals with deficiency in FXII have a normal hemostatic capacity, animal models revealed a critical role of FXIIa-driven coagulation in thromboembolic diseases. In addition to its role in thrombosis, FXIIa contributes to inflammation through the activation of the inflammatory bradykinin-producing kallikrein-kinin system. Pharmacological inhibition of FXII/FXIIa interferes with thrombosis and inflammation in animal models. Thus, targeting the FXIIa-driven contact system seems to be a promising and safe therapeutic anticoagulation treatment strategy, with additional anti-inflammatory effects. Here, we discuss novel functions of FXIIa in cardiovascular thrombotic and inflammatory disorders.

The polyphosphate-factor XII pathway drives coagulation in prostate cancer-associated thrombosis.

Cancer is a leading cause of thrombosis. We identify a new procoagulant mechanism that contributes to thromboembolism in prostate cancer and allows for safe anticoagulation therapy development. Prostate cancer-mediated procoagulant activity was reduced in plasma in the absence of factor XII or its substrate of the intrinsic coagulation pathway factor XI. Prostate cancer cells and secreted prostasomes expose long chain polyphosphate on their surface that colocalized with active factor XII and initiated coagulation in a factor XII-dependent manner. Polyphosphate content correlated with the procoagulant activity of prostasomes. Inherited deficiency in factor XI or XII or high-molecular-weight kininogen, but not plasma kallikrein, protected mice from prostasome-induced lethal pulmonary embolism. Targeting polyphosphate or factor XII conferred resistance to prostate cancer-driven thrombosis in mice, without increasing bleeding. Inhibition of factor XII with recombinant 3F7 antibody reduced the increased prostasome-mediated procoagulant activity in patient plasma. The data illustrate a critical role for polyphosphate/factor XII-triggered coagulation in prostate cancer-associated thrombosis with implications for anticoagulation without therapy-associated bleeding in malignancies.

A Synthetic Factor XIIa Inhibitor Blocks Selectively Intrinsic Coagulation Initiation.

Coagulation factor XII (FXII) inhibitors are of interest for the study of the protease in the intrinsic coagulation pathway, for the suppression of contact activation in blood coagulation assays, and they have potential application in antithrombotic therapy. However, synthetic FXII inhibitors developed to date have weak binding affinity and/or poor selectivity. Herein, we developed a peptide macrocycle that inhibits activated FXII (FXIIa) with an inhibitory constant Ki of 22 nM and a selectivity of >2000-fold over other proteases. Sequence and structure analysis revealed that one of the two macrocyclic rings of the in vitro evolved peptide mimics the combining loop of corn trypsin inhibitor, a natural protein-based inhibitor of FXIIa. The synthetic inhibitor blocked intrinsic coagulation initiation without affecting extrinsic coagulation. Furthermore, the peptide macrocycle efficiently suppressed plasma coagulation triggered by contact of blood with sample tubes and allowed specific investigation of tissue factor initiated coagulation.

Ayadualin, a novel RGD peptide with dual antihemostatic activities from the sand fly Lutzomyia ayacuchensis, a vector of Andean-type cutaneous leishmaniasis.

Sequence analysis of the Lutzomyia (Lu.) ayacuchensis salivary gland cDNA library identified a short peptide containing an RGD (Arg-Gly-Asp) sequence flanked by two cysteine residues in the C-terminal end as the most abundant transcript. In the present study, a recombinant protein of the RGD-containing peptide, designated ayadualin, was expressed in Escherichia coli and its activity was characterized. Ayadualin inhibited both collagen and ADP-induced platelet aggregations by interfering with the binding of integrin αIIbß3 to fibrinogen. The RGD sequence and cysteine residues located on both sides of the RGD sequence were essential for the inhibitory action. Moreover, ayadualin efficiently inhibited the intrinsic blood coagulation pathway irrespective of the RGD sequence. Measuring the enzymatic activity of coagulation factors using chromogenic substrates revealed that ayadualin efficiently inhibited factor XIIa (FXIIa) activity in a dose-dependent manner. In addition, pre-incubation of ayadualin with FXII inhibited FXIIa activity, while activated FXIIa was not affected by ayadualin, indicating that ayadualin inhibits the activation of FXII, but not enzymatic activity of FXIIa. These results indicated that ayadualin plays an important role in the blood feeding of Lu. ayacuchensis by inhibiting host hemostasis via dual mechanisms.

Excess of heme induces tissue factor-dependent activation of coagulation in mice.

An excess of free heme is present in the blood during many types of hemolytic anemia. This has been linked to organ damage caused by heme-mediated oxidative stress and vascular inflammation. We investigated the mechanism of heme-induced coagulation activation in vivo. Heme caused coagulation activation in wild-type mice that was attenuated by an anti-tissue factor antibody and in mice expressing low levels of tissue factor. In contrast, neither factor XI deletion nor inhibition of factor XIIa-mediated factor XI activation reduced heme-induced coagulation activation, suggesting that the intrinsic coagulation pathway is not involved. We investigated the source of tissue factor in heme-induced coagulation activation. Heme increased the procoagulant activity of mouse macrophages and human PBMCs. Tissue factor-positive staining was observed on leukocytes isolated from the blood of heme-treated mice but not on endothelial cells in the lungs. Furthermore, heme increased vascular permeability in the mouse lungs, kidney and heart. Deletion of tissue factor from either myeloid cells, hematopoietic or endothelial cells, or inhibition of tissue factor expressed by non-hematopoietic cells did not reduce heme-induced coagulation activation. However, heme-induced activation of coagulation was abolished when both non-hematopoietic and hematopoietic cell tissue factor was inhibited. Finally, we demonstrated that coagulation activation was partially attenuated in sickle cell mice treated with recombinant hemopexin to neutralize free heme. Our results indicate that heme promotes tissue factor-dependent coagulation activation and induces tissue factor expression on leukocytes in vivo. We also demonstrated that free heme may contribute to thrombin generation in a mouse model of sickle cell disease.

Novel family of insect salivary inhibitors blocks contact pathway activation by binding to polyphosphate, heparin, and dextran sulfate.

OBJECTIVE: Polyphosphate and heparin are anionic polymers released by activated mast cells and platelets that are known to stimulate the contact pathway of coagulation. These polymers promote both the autoactivation of factor XII and the assembly of complexes containing factor XI, prekallikrein, and high-molecular-weight kininogen. We are searching for salivary proteins from blood-feeding insects that counteract the effect of procoagulant and proinflammatory factors in the host, including elements of the contact pathway. APPROACH AND RESULTS: Here, we evaluate the ability of the sand fly salivary proteins, PdSP15a and PdSP15b, to inhibit the contact pathway by disrupting binding of its components to anionic polymers. We attempt to demonstrate binding of the proteins to polyphosphate, heparin, and dextran sulfate. We also evaluate the effect of this binding on contact pathway reactions. We also set out to determine the x-ray crystal structure of PdSP15b and examine the determinants of relevant molecular interactions. Both proteins bind polyphosphate, heparin, and dextran sulfate with high affinity. Through this mechanism they inhibit the autoactivation of factor XII and factor XI, the reciprocal activation of factor XII and prekallikrein, the activation of factor XI by thrombin and factor XIIa, the cleavage of high-molecular-weight kininogen in plasma, and plasma extravasation induced by polyphosphate. The crystal structure of PdSP15b contains an amphipathic helix studded with basic side chains that forms the likely interaction surface. CONCLUSIONS: The results of these studies indicate that the binding of anionic polymers by salivary proteins is used by blood feeders as an antihemostatic/anti-inflammatory mechanism.

Factor XII promotes blood coagulation independent of factor XI in the presence of long-chain polyphosphates.

BACKGROUND: Inorganic polyphosphates (polyP), which are secreted by activated platelets (short-chain polyP) and accumulate in some bacteria (long-chain polyP), support the contact activation of factor XII (FXII) and accelerate the activation of FXI. OBJECTIVES: The aim of the present study was to evaluate the role of FXI in polyP-mediated coagulation activation and experimental thrombus formation. METHODS AND RESULTS: Pretreatment of plasma with antibodies that selectively inhibit FXI activation by activated FXII (FXIIa) or FIX) activation by activated FXI (FXIa) were not able to inhibit the procoagulant effect of long or short-chain polyP in plasma. In contrast, the FXIIa inhibitor, corn trypsin inhibitor, blocked the procoagulant effect of long and short polyP in plasma. In a purified system, long polyP significantly enhanced the rate of FXII and prekallikrein activation and the activation of FXI by thrombin but not by FXIIa. In FXI-deficient plasma, long polyP promoted clotting of plasma in an FIX-dependent manner. In a purified system, the activation of FXII and prekallikrein by long polyP promoted FIX activation and prothombin activation. In an ex vivo model of occlusive thrombus formation, inhibition of FXIIa with corn trypsin inhibitor but not of FXI with a neutralizing antibodies abolished the prothrombotic effect of long polyP. CONCLUSIONS: We propose that long polyP promotes FXII-mediated blood coagulation bypassing FXI. Accordingly, some polyp-containing pathogens may have evolved strategies to exploit polyP-initiated FXII activation for virulence, and selective inhibition of FXII may improve the host response to pathogens.

A proteinase inhibitor from Caesalpinia echinata (pau-brasil) seeds for plasma kallikrein, plasmin and factor XIIa.

Caesalpinia echinata is a tree belonging to the Leguminosae family. The red color of the trunk, looking like burning wood ('brasa' in Portuguese), is the origin of the name Brazil. Seeds of leguminous plants contain high amounts of serine proteinase inhibitors that can affect different biological processes. Here we show that a protein isolated from seeds of C. echinata is able to inhibit enzymes that participate in blood coagulation and fibrinolysis. This inhibitor (CeKI) was purified to homogeneity by ion exchange and reversed-phase chromatography. SDS-PAGE indicated a single polypeptide chain with a molecular mass of 20 kDa. CeKI inhibits human plasma kallikrein ( K i =3.1 nM), plasmin ( K i =0.18 nM), factor XIIa ( K i =0.18 nM), trypsin ( K i =21.5 nM) and factor Xa ( K i =0.49 mM). CeKI inhibited kinin release from highmolecular- mass kininogen by kallikrein in vitro . The N-terminal sequence, determined by automatic Edman degradation, identified the inhibitor as a member of the Kunitz family. The secondary structure, determined by circular dichroism, is mainly a random coil followed by beta-sheet structure. The action of CeKI on enzymes of the blood-clotting intrinsic pathway was confirmed by prolongation of the activated partial thromboplastin time.

Unusual proteolytic activation of pro-hepatocyte growth factor by plasma kallikrein and coagulation factor XIa.

Hepatocyte growth factor (HGF), the ligand for the receptor tyrosine kinase c-Met, is composed of an alpha-chain containing four Kringle domains (K1-K4) and a serine protease domain-like beta-chain. Receptor activation by HGF is contingent upon prior proteolytic conversion of the secreted inactive single chain form (pro-HGF) into the biologically active two chain form by a single cleavage at the Arg(494)-Val(495) bond. By screening a panel of serine proteases we identified two new HGF activators, plasma kallikrein and coagulation factor XIa (FXIa). The concentrations of kallikrein and FXIa to cleave 50% (EC(50)) of (125)I-labeled pro-HGF during a 4-h period were 10 and 17 nm. Unlike other known activators, both FXIa and kallikrein processed pro-HGF by cleavage at two sites. Using N-terminal sequencing they were identified as the normal cleavage site Arg(494)-Val(495) and the novel site Arg(424)-His(425) located in the K4 domain of the alpha-chain. The identity of this unusual second cleavage site was firmly established by use of the double mutant HGF(R424A/R494E), which was completely resistant to cleavage by kallikrein and FXIa. Experiments with another mutant form, HGF(Arg(494) --> Glu), indicated that cleavage at the K4 site was independent of a prior cleavage at the primary, kinetically preferred Arg(494)-Val(495) site. The cleavage at the K4 site had no obvious consequences on HGF function, because it was fully capable of phosphorylating the c-Met receptor of A549 cells. This may be explained by the disulfide bond network in K4, which holds the cleaved alpha-chain together. In conclusion, the ability of plasma kallikrein and FXIa to activate pro-HGF in vitro raises the possibility that mediators of inflammation and blood coagulation may also regulate processes that involve the HGF/c-Met pathway, such as tissue repair and angiogenesis.

The lack of augmentation by aspirin of inhibition of platelet reactivity by ticlopidine.

A decreased threshold for platelet activation apparently contributes to the risk of cardiovascular events, such as acute myocardial infarction. To evaluate the impact of specific agents, we characterized platelet reactivity in 9 healthy subjects before and after 5 days of ingestion of 4 commonly prescribed regimens, 81 mg of aspirin daily, 325 mg of aspirin daily, ticlopidine 250 mg twice daily, and ticlopidine plus 325 mg of aspirin daily. Platelet reactivity was assessed with (1) aggregometry induced by 4 microM adenosine diphosphate (ADP) and collagen (0.19 mg/ml) and performed in platelet-rich plasma; and (2) flow cytometric determination of ADP-induced (0.2, 0.8, and 1.5 microM) P-selectin expression in whole blood. Because anticoagulants alter platelet reactivity, results were obtained with 3 anticoagulants, citrate, enoxaparin, or corn trypsin inhibitor (CTI, a specific inhibitor of factor XIIa without effect on other coagulation factors). Ingestion of aspirin did not alter platelet activation as assessed with flow cytometry. Inhibition of the second phase of aggregation was seen with ADP-induced aggregation in platelet-rich plasma anticoagulated with citrate but not enoxaparin or CTI. Ingestion of ticlopidine led to inhibition of ADP-induced aggregation and P-selectin expression. Inhibition of platelet reactivity after the combination of aspirin and ticlopidine did not differ from ticlopidine alone. Marked interindividual variability in platelet reactivity was seen after ingestion of ticlopidine. The results indicate that assessment of effects of specific pharmacologic regimens with accurate and readily available assays of platelet reactivity may facilitate effective prophylaxis and treatment of high-risk subjects with antiplatelet regimens designed to optimally diminish platelet reactivity.

Tridegin, a novel peptidic inhibitor of factor XIIIa from the leech, Haementeria ghilianii, enhances fibrinolysis in vitro.

Tridegin is a potent inhibitor of factor XIIIa from the leech, Haementeria ghilianii, which inhibits protein cross-linking. It modifies plasmin-mediated fibrin degradation as shown by the absence of D-dimer and approximately halves the time for fibrinolysis. Plasma clots formed in the presence of Tridegin lyse more rapidly when either streptokinase, tissue plasminogen activator or hementin is added 2 h after clot formation. The effect of Tridegin is markedly increased if clots are formed from platelet-rich plasma. Platelet-rich plasma clots are lysed much more slowly by the fibrinolytic enzymes used and if Tridegin is present, the rate of lysis returns almost to that of platelet-free clots. These studies indicate the important role of platelets in conferring resistance to commonly used fibrinolytic enzymes and suggest that protein cross-linking is an important step in this effect. Moreover they indicate that Tridegin, a small polypeptide, may have potential as an adjunct to thrombolytic therapy.

Hepatocyte growth factor activator inhibitor, a novel Kunitz-type serine protease inhibitor.

Hepatocyte growth factor (HGF) activator is a serine protease that is produced and secreted by the liver and circulates in the blood as an inactive zymogen. In response to tissue injury, the HGF activator zymogen is converted to the active form by limited proteolysis. The activated HGF activator converts an inactive single chain precursor of HGF to a biologically active heterodimer in injured tissue. The activated HGF may be involved in the regeneration of the injured tissue. In this study, we purified an inhibitor of HGF activator from the conditioned medium of a human MKN45 stomach carcinoma cell line and molecularly cloned its cDNA. The sequence of the cDNA revealed that the inhibitor has two well defined Kunitz domains, suggesting that the inhibitor is a member of the Kunitz family of serine protease inhibitors. The sequence also showed that the primary translation product of the inhibitor has a hydrophobic sequence at the COOH-terminal region. Inhibitory activity toward HGF activator was detected in the membrane fraction as well as in the conditioned medium of MKN45 cells. These results suggest that the inhibitor may be produced as a membrane-associated form and secreted by the producing cells as a proteolytically truncated form.

Changes in the contact system during orthotopic liver transplantation with and without aprotinin.

The main cause of nonsurgical bleeding during orthotopic liver transplantation has been attributed to be hyperfibrinolysis due to high plasma levels of tissue plasminogen activator. The aim of this study was to investigate contact activation and its possible contribution to fibrinolysis during OLT with and without aprotinin. Aprotinin or placebo was given to 20 patients undergoing OLT as part of a randomized double-blind trial. Plasma samples were collected before, during, and after OLT. There were decreased preoperative levels of prekallikrein and factor XIIa (P < 0.05), with a trend for kallikrein and factor XIIa activity to increase during OLT peaking on reperfusion (P < 0.05). Kallikrein inhibition, C1 esterase inhibitor, and alpha-2-macroglobulin levels were normal before surgery, with low normal levels of antithrombin III and alpha-2-antiplasmin; these levels decreased during OLT with no specific change on reperfusion. In the aprotinin-treated group, kallikrein inhibition levels increased (P < 0.05) from preoperative mean (+/- SD) values of 101 +/- 47% to 154 +/- 42% and antiplasmin levels increased (P < 0.05) from 72 +/- 28% to 243 +/- 53% during the anhepatic phase, reflecting the effect of aprotinin. The antifibrinolytic effect of aprotinin was demonstrated by decreased levels of D-dimer on reperfusion (P < 0.05) and at the end of OLT (P < 0.001) in the aprotinin-treated group. We have shown that contact activation during OLT is minimal and that aprotinin does not alter the pattern of contact activation, but provides an antikallikrein effect.

Aprotinin in cardiopulmonary bypass--effects on the Hageman factor (FXII)--Kallikrein system and blood loss.

Aprotinin has been used in our hospital in open heart surgery for almost 20 years and recently published studies have revealed a reduction in postoperative blood loss under this therapy. In the present study patients undergoing aorto-coronary bypass operations received either 20,000 KIU aprotinin/kg body weight (group 2) or 60,000 KIU aprotinin/kg body weight (group 3). Another group of patients without aprotinin served as a control (group 1) and postoperative bleeding was more pronounced in these patients compared with the other groups. In parallel, slight reductions in kallikrein-like activity were observed in patients treated with aprotinin. Furthermore, we have shown that the main inhibitor of the contact phase, C1-esterase-inhibitor, loses some of its activity against beta-FXIIa in the presence of heparin. Aprotinin was able to partly antagonize this phenomenon. All studies dealing with the effect of aprotinin in extracorporeal circulation demonstrate hyperfibrinolysis in untreated patients. Aprotinin is known to inhibit plasmin at low concentrations and thus reduced the postoperative bleeding tendency (group 2). In addition, plasma kallikrein is inhibited by high aprotinin concentrations and is responsible for a reduced activation of the contact phase system. This effect led to a further reduction in blood loss (group 3).