Role of thrombin in coronary artery bypass grafting

Thrombin is a multifunctional protease, which has a central role in the development and progression of coronary atherosclerotic lesions and it is a possible mediator of myocardial ischemia-reperfusion injury. Its generation and procoagulant activity are greatly upregulated during cardiopulmonary bypass (CPB). On the other hand, activated protein C, a physiologic anticoagulant that is activated by thrombomodulin-bound thrombin, has been beneficial in various models of ischemia-reperfusion. Therefore, our aim in this study was to test whether thrombin generation or protein C activation during coronary artery bypass grafting (CABG) associate with postoperative myocardial damage or hemodynamic changes. To further investigate the regulation of thrombin during CABG, we tested whether preoperative thrombophilic factors associate with increased CPB-related generation of thrombin or its procoagulant activity. We also measured the anticoagulant effects of heparin during CPB with a novel coagulation test, prothrombinase-induced clotting time (PiCT), and compared the performance of this test with the present standard of laboratory-based anticoagulation monitoring. One hundred patients undergoing elective on-pump CABG were studied prospectively. A progressive increase in markers of thrombin generation (F1+2), fibrinolysis (D-dimer), and fibrin formation (soluble fibrin monomer complexes) was observed during CPB, which was further distinctly propagated by reperfusion after myocardial ischemia, and continued to peak after the neutralization of heparin with protamine. Thrombin generation during reperfusion after CABG associated with postoperative myocardial damage and increased pulmonary vascular resistance. Activated protein C levels increased only slightly during CPB before the release of the aortic clamp, but reperfusion and more significantly heparin neutralization caused a massive increase in activated protein C levels. Protein C activation was clearly delayed in relation to both thrombin generation and fibrin formation. Even though activated protein C associated dynamically with postoperative hemodynamic performance, it did not associate with postoperative myocardial damage. Preoperative thrombophilic variables did not associate with perioperative thrombin generation or its procoagulant activity. Therefore, our results do not favor routine thrombophilia screening before CABG. There was poor agreement between PiCT and other measurements of heparin effects in the setting of CPB. However, lower heparin levels during CPB associated with inferior thrombin control and high heparin levels during CPB associated with fewer perioperative transfusions of blood products. Overall, our results suggest that hypercoagulation after CABG, especially during reperfusion, might be clinically important.

Cardiac function before and after treatment for various types of loading conditions and in myocardial restriction : A prospective study on pediatric patients with two- and three-dimensional echocardiography and measurement of serum natriuretic peptides

Background: The incidence of all forms of congenital heart defects is 0.75%. For patients with congenital heart defects, life-expectancy has improved with new treatment modalities. Structural heart defects may require surgical or catheter treatment which may be corrective or palliative. Even those with corrective therapy need regular follow-up due to residual lesions, late sequelae, and possible complications after interventions. Aims: The aim of this thesis was to evaluate cardiac function before and after treatment for volume overload of the right ventricle (RV) caused by atrial septal defect (ASD), volume overload of the left ventricle (LV) caused by patent ductus arteriosus (PDA), and pressure overload of the LV caused by coarctation of the aorta (CoA), and to evaluate cardiac function in patients with Mulibrey nanism. Methods: In Study I, of the 24 children with ASD, 7 underwent surgical correction and 17 percutaneous occlusion of ASD. Study II had 33 patients with PDA undergoing percutaneous occlusion. In Study III, 28 patients with CoA underwent either surgical correction or percutaneous balloon dilatation of CoA. Study IV comprised 26 children with Mulibrey nanism. A total of 76 healthy voluntary children were examined as a control group. In each study, controls were matched to patients. All patients and controls underwent clinical cardiovascular examinations, two-dimensional (2D) and three-dimensional (3D) echocardiographic examinations, and blood sampling for measurement of natriuretic peptides prior to the intervention and twice or three times thereafter. Control children were examined once by 2D and 3D echocardiography. M-mode echocardiography was performed from the parasternal long axis view directed by 2D echocardiography. The left atrium-to-aorta (LA/Ao) ratio was calculated as an index of LA size. The end-diastolic and end-systolic dimensions of LV as well as the end-diastolic thicknesses of the interventricular septum and LV posterior wall were measured. LV volumes, and the fractional shortening (FS) and ejection fraction (EF) as indices of contractility were then calculated, and the z scores of LV dimensions determined. Diastolic function of LV was estimated from the mitral inflow signal obtained by Doppler echocardiography. In three-dimensional echocardiography, time-volume curves were used to determine end-diastolic and end-systolic volumes, stroke volume, and EF. Diastolic and systolic function of LV was estimated from the calculated first derivatives of these curves. Results: (I): In all children with ASD, during the one-year follow-up, the z score of the RV end-diastolic diameter decreased and that of LV increased. However, dilatation of RV did not resolve entirely during the follow-up in either treatment group. In addition, the size of LV increased more slowly in the surgical subgroup but reached control levels in both groups. Concentrations of natriuretic peptides in patients treated percutaneously increased during the first month after ASD closure and normalized thereafter, but in patients treated surgically, they remained higher than in controls. (II): In the PDA group, at baseline, the end-diastolic diameter of LV measured over 2SD in 5 of 33 patients. The median N-terminal pro-brain natriuretic peptide (proBNP) concentration before closure measured 72 ng/l in the control group and 141 ng/l in the PDA group (P = 0.001) and 6 months after closure measured 78.5 ng/l (P = NS). Patients differed from control subjects in indices of LV diastolic and systolic function at baseline, but by the end of follow-up, all these differences had disappeared. Even in the subgroup of patients with normal-sized LV at baseline, the LV end-diastolic volume decreased significantly during follow-up. (III): Before repair, the size and wall thickness of LV were higher in patients with CoA than in controls. Systolic blood pressure measured a median 123 mm Hg in patients before repair (P < 0.001) and 103 mm Hg one year thereafter, and 101 mm Hg in controls. The diameter of the coarctation segment measured a median 3.0 mm at baseline, and 7.9 at the 12-month (P = 0.006) follow-up. Thicknesses of the interventricular septum and posterior wall of the LV decreased after repair but increased to the initial level one year thereafter. The velocity time integrals of mitral inflow increased, but no changes were evident in LV dimensions or contractility. During follow-up, serum levels of natriuretic peptides decreased correlating with diastolic and systolic indices of LV function in 2D and 3D echocardiography. (IV): In 2D echocardiography, the interventricular septum and LV posterior wall were thicker, and velocity time integrals of mitral inflow shorter in patients with Mulibrey nanism than in controls. In 3D echocardiography, LV end-diastolic volume measured a median 51.9 (range 33.3 to 73.4) ml/m² in patients and 59.7 (range 37.6 to 87.6) ml/m² in controls (P = 0.040), and serum levels of ANPN and proBNP a median 0.54 (range 0.04 to 4.7) nmol/l and 289 (range 18 to 9170) ng/l, in patients and 0.28 (range 0.09 to 0.72) nmol/l (P < 0.001) and 54 (range 26 to 139) ng/l (P < 0.001) in controls. They correlated with several indices of diastolic LV function. Conclusions (I): During the one-year follow-up after the ASD closure, RV size decreased but did not normalize in all patients. The size of the LV normalized after ASD closure but the increase in LV size was slower in patients treated surgically than in those treated with the percutaneous technique. Serum levels of ANPN and proBNP were elevated prior to ASD closure but decreased thereafter to control levels in patients treated with the percutaneous technique but not in those treated surgically. (II): Changes in LV volume and function caused by PDA disappeared by 6 months after percutaneous closure. Even the children with normal-sized LV benefited from the procedure. (III): After repair of CoA, the RV size and the velocity time integrals of mitral inflow increased, and serum levels of natriuretic peptides decreased. Patients need close follow-up, despite cessation of LV pressure overload, since LV hypertrophy persisted even in normotensive patients with normal growth of the coarctation segment. (IV): In children with Mulibrey nanism, the LV wall was hypertrophied, with myocardial restriction and impairment of LV function. Significant correlations appeared between indices of LV function, size of the left atrium, and levels of natriuretic peptides, indicating that measurement of serum levels of natriuretic peptides can be used in the clinical follow-up of this patient group despite its dependence on loading conditions.

Inhibition of Thrombin in Cardiac Surgery : experiments in a porcine model

Cardiac surgery involving cardiopulmonary bypass (CPB) induces activation of inflammation and coagulation systems and is associated with ischemia-reperfusion injury (I/R injury)in various organs including the myocardium, lungs, and intestine. I/R injury is manifested as organ dysfunction. Thrombin, the key enzyme of coagulation , plays a cenral role also in inflammation and contributes to regulation of apoptosis as well. The general aim of this thesis was to evaluate the potential of thrombin inhibition in reducing the adverse effects of I/R injury in myocardium, lungs, and intestine associated with the use of CPB and cardiac surgery. Forty five pigs were used for the studies. Two randomized blinded studies were performed. Animals underwent 75 min of normothermic CPB, 60 min of aortic clamping, and 120 min of reperfusion period. Twenty animals received iv. recombinant hirudin, a selective and effective inbitor of thrombin, or placebo. In a similar setting, twenty animals received an iv-bolus (250 IU/kg) of antithrombin (AT) or placebo. An additional group of 5 animals received 500 IU/kg in an open label setting to test dose response. Generation of thrombin (TAT), coagulation status (ACT), and hemodynamics were measured. Intramucosal pH and pCO2 were measured from the luminal surface of ileum using tonometry simultaneusly with arterial gas analysis. In addition, myocardial, lung, and intestinal biopsies were taken to quantitate leukocyte infiltration (MPO), for histological evaluation, and detection of apoptosis (TUNEL, caspase 3). In conclusion, our data suggest that r-hirudin may be an effective inhibitor of reperfusion induced thrombin generation in addition to being a direct inhibitor of preformed thrombin. Overall, the results suggest that inhibition of thrombin, beyond what is needed for efficient anticoagulation by heparin, has beneficial effects on myocardial I/R injury and hemodynamics during cardiac surgery and CPB. We showed that infusion of the thrombin inhibitor r-hirudin during reperfusion was associated with attenuated post ischemia left ventricular dysfunction and decreased systemic vascular resistance. Consequently microvascular flow was improved during ischemia-reperfusion injury. Improved recovery of myocardium during the post-ischemic reperfusion period was associated with significantly less cardiomyocyte apoptosis and with a trend in anti-inflammatory effects. Thus, inhibition of reperfusion induced thrombin may offer beneficial effects by mechanisms other than direct anticoagulant effects. AT, in doses with a significant anticoagulant effect, did not alleviate myocardial I/R injury in terms of myocardial recovery, histological inflammatory changes or post-ischemic troponin T release. Instead, AT attenuated reperfusion induced increase in pulmonary pressure after CPB. Taken the clinical significance of postoperative pulmonary hemodynamics in patients undergoing cardiopulmonary bypass, the potential positive regulatory role of AT and clinical implications needs to be studied further. Inflammatory response in the gut wall proved to be poorly associated with perturbed mucosal perfusion and the animals with the least neutrophil tissue sequestration and I/R related histological alterations tended to have the most progressive mucosal hypoperfusion. Thus, mechanisms of low-flow reperfusion injury during CPB can differ from the mechanisms seen in total ischemia reperfusion injury.

Electrocardiographic repolarization variables in detecting myocardial infarction and ischemic injury : From body surface potential mapping to a single lead

The aim of the studies was to improve the diagnostic capability of electrocardiography (ECG) in detecting myocardial ischemic injury with a future goal of an automatic screening and monitoring method for ischemic heart disease. The method of choice was body surface potential mapping (BSPM), containing numerous leads, with intention to find the optimal recording sites and optimal ECG variables for ischemia and myocardial infarction (MI) diagnostics. The studies included 144 patients with prior MI, 79 patients with evolving ischemia, 42 patients with left ventricular hypertrophy (LVH), and 84 healthy controls. Study I examined the depolarization wave in prior MI with respect to MI location. Studies II-V examined the depolarization and repolarization waves in prior MI detection with respect to the Minnesota code, Q-wave status, and study V also with respect to MI location. In study VI the depolarization and repolarization variables were examined in 79 patients in the face of evolving myocardial ischemia and ischemic injury. When analyzed from a single lead at any recording site the results revealed superiority of the repolarization variables over the depolarization variables and over the conventional 12-lead ECG methods, both in the detection of prior MI and evolving ischemic injury. The QT integral, covering both depolarization and repolarization, appeared indifferent to the Q-wave status, the time elapsed from MI, or the MI or ischemia location. In the face of evolving ischemic injury the performance of the QT integral was not hampered even by underlying LVH. The examined depolarization and repolarization variables were effective when recorded in a single site, in contrast to the conventional 12-lead ECG criteria. The inverse spatial correlation of the depolarization and depolarization waves in myocardial ischemia and injury could be reduced into the QT integral variable recorded in a single site on the left flank. In conclusion, the QT integral variable, detectable in a single lead, with optimal recording site on the left flank, was able to detect prior MI and evolving ischemic injury more effectively than the conventional ECG markers. The QT integral, in a single-lead or a small number of leads, offers potential for automated screening of ischemic heart disease, acute ischemia monitoring and therapeutic decision-guiding as well as risk stratification.

Thrombin regulation in newborn infants

The coagulation system of newborn infants differs markedly from that of older children and adults. The activities of most coagulation factors and anticoagulants are low, leading to altered regulation in the formation of the key enzyme, thrombin. Timely and adequate generation of thrombin is essential, as thrombin activates platelets and many coagulation factors, cleaves fibrinogen into fibrin and activates the antithrombotic and anti-inflammatory protein C pathway. On the other hand, excess thrombin may promote thrombotic complications and exacerbate harmful inflammatory reactions. Despite the characteristic features, the newborn coagulation system can be considered physiological, since healthy newborns rarely show haemorrhagic or thrombotic complications. Sick newborns, however, often encounter clinical situations that challenge their coagulation system. The aim of this study was to clarify the behaviour of the neonatal coagulation system in selected clinical situations, with a special emphasis on the generation of thrombin. Thrombin was measured by in vivo thrombin generation markers and by thrombin generation potential in vitro. The patient groups included sick newborns undergoing intensive care and receiving fresh-frozen plasma (FFP), requiring exchange transfusions (ET) or presenting with a congenital heart defect requiring open heart surgery. Additionally, healthy newborns with inherited heterozygous factor V Leiden (FVL) mutation were studied. Thrombin generation potential was also analysed in cord plasma of healthy infants and in adults. Healthy as well as sick newborn infants showed lower total thrombin generation potential in vitro but faster initiation of thrombin generation than adults. These findings were qualitatively similar when plasma was supplemented with platelets. Platelets, however, significantly altered the effect of the major anticoagulant, activated protein C (APC), on thrombin generation potential. In accordance with previous studies, thrombin generation in healthy newborn platelet-poor plasma was resistant to the anticoagulant effects of APC, but when the plasma was supplemented with platelets APC attenuated thrombin generation significantly more in newborns than in adults. In vivo generation of thrombin was elevated in nearly all of the sick newborn infants. The low-volume FFP transfusion as opposed to the change from neonatal to adult blood in ET exerted markedly different effects on neonatal thrombin generation. FFP reduced the in vivo generation of thrombin in those newborns with the highest pretransfusional thrombin generation, thus acting as an anticoagulant agent. In those infants with lower pretransfusional thrombin generation, the effect of FFP on thrombin generation was fairly neutral. On the other hand, the combination of red blood cells and FFP, used to perform ET, significantly increased the in vivo thrombin formation and shifted the balance in the newborn coagulation system to the procoagulant direction. Cardiopulmonary bypass (CPB) also significantly increased the in vivo thrombin generation, but the thrombin generation profile during CPB differed from that previously observed in adults. Escalation of thrombin at early reperfusion was not observed in newborns; in adults, its occurrence is associated with postoperative myocardial damage. Finally, in healthy newborns with FVL heterozygosity, faster initiation of thrombin generation was observed compared with controls. Interestingly, FV level was lower in FVL-heterozygous infants, possibly to counteract the procoagulant effects induced by FVL. In conclusion, unique features regarding thrombin regulation in newborn infants were observed. These features included a novel platelet effect on the regulation of the protein C pathway. The clinical challenges mainly seemed to shift the balance in the coagulation system of newborns to the procoagulant direction. Blood component transfusions markedly affected coagulation in a manner specific to the product but that could also be altered by the clinical situation. Overall, the results highlight the need for understanding developmental haemostasis for both diagnostic and therapeutic purposes.

Ischemia-reperfusion injury in human liver transplantation : Mechanisms and effects on graft function

Liver transplantation is an established therapy for both acute and chronic liver failure. Despite excellent long-term outcome, graft dysfunction remains a problem affecting up to 15-30% of the recipients. The etiology of dysfunction is multifactorial, with ischemia-reperfusion injury regarded as one of the most important contributors. This thesis focuses on the inflammatory response during graft procurement and reperfusion in liver transplantation in adults. Activation of protein C was examined as a potential endogenous anti-inflammatory mechanism. The effects of inflammatory responses on graft function and outcome were investigated. Seventy adult patients undergoing liver transplantation in Helsinki University Central Hospital, and 50 multiorgan donors, were studied. Blood samples from the portal and the hepatic veins were drawn before graft procurement and at several time points during graft reperfusion to assess changes within the liver. Liver biopsies were taken before graft preservation and after reperfusion. Neutrophil and monocyte CD11b and L-selectin expression were analysed by flow cytometry. Plasma TNF-α, IL-6, IL-8, sICAM-1, and HMGB1 were determined by ELISA and Western-blotting. HMGB1 immunohistochemistry was performed on liver tissue specimens. Plasma protein C and activated protein C were determined by an enzyme-capture assay. Hepatic IL-8 release during graft procurement was associated with subsequent graft dysfunction, biliary in particular, in the recipient. Biliary marker levels increased only 5 7 days after transplantation. Thus, donor inflammatory response appears to influence recipient liver function with relatively long-lasting effects. Hepatic phagocyte activation and sequestration, with concomitant HMGB1 release, occurred during reperfusion. Neither phagocyte activation nor plasma cytokines correlated with postoperative graft function. Thus, activation of the inflammatory responses within the liver during reperfusion may be of minor clinical significance. However, HMGB1 was released from hepatocytes and were also correlated with postoperative transaminase levels. Accordingly, HMGB1 appears to be a marker of hepatocellular injury.

Heme oxygenase-1 in cardiovascular diseases

Myocardial infarction (MI) and heart failure are major causes of morbidity and mortality worldwide. Treatment of MI involves early restoration of blood flow to limit infarct size and preserve cardiac function. MI leads to left ventricular remodeling, which may eventually progress to heart failure, despite the established pharmacological treatment of the disease. To improve outcome of MI, new strategies for protecting the myocardium against ischemic injury and enhancing the recovery and repair of the infarcted heart are needed. Heme oxygenase-1 (HO-1) is a stress-responsive and cytoprotective enzyme catalyzing the degradation of heme into the biologically active reaction products biliverdin/bilirubin, carbon monoxide (CO) and free iron. HO-1 plays a key role in maintaining cellular homeostasis by its antiapoptotic, anti-inflammatory, antioxidative and proangiogenic properties. The present study aimed, first, at evaluating the role of HO-1 as a cardioprotective and prohealing enzyme in experimental rat models and at investigating the potential mechanisms mediating the beneficial effects of HO-1 in the heart. The second aim was to evaluate the role of HO-1 in 231 critically ill intensive care unit (ICU) patients by investigating the association of HO-1 polymorphisms and HO-1 plasma concentrations with illness severity, organ dysfunction and mortality throughout the study population and in the subgroup of cardiac patients. We observed in an experimental rat MI model, that HO-1 expression was induced in the infarcted rat hearts, especially in the infarct and infarct border areas. In addition, pre-emptive HO-1 induction and CO donor pretreatment promoted recovery and repair of the infarcted hearts by differential mechanisms. CO promoted vasculogenesis and formation of new cardiomyocytes by activating c-kit+ stem/progenitor cells via hypoxia-inducible factor 1 alpha, stromal cell-derived factor 1 alpha (SDF-1a) and vascular endothelial growth factor B, whereas HO-1 promoted angiogenesis possibly via SDF-1a. Furthermore, HO-1 protected the heart in the early phase of infarct healing by increasing survival and proliferation of cardiomyocytes. The antiapoptotic effect of HO-1 persisted in the late phases of infarct healing. HO-1 also modulated the production of extracellular matrix components and reduced perivascular fibrosis. Some of these beneficial effects of HO-1 were mediated by CO, e.g. the antiapoptotic effect. However, CO may also have adverse effects on the heart, since it increased the expression of extracellular matrix components. In isolated perfused rat hearts, HO-1 induction improved the recovery of postischemic cardiac function and abrogated reperfusion-induced ventricular fibrillation, possibly in part via connexin 43. We found that HO-1 plasma levels were increased in all critically ill patients, including cardiac patients, and were associated with the degree of organ dysfunction and disease severity. HO-1 plasma concentrations were also higher in ICU and hospital nonsurvivors than in survivors, and the maximum HO-1 concentration was an independent predictor of hospital mortality. Patients with the HO-1 -413T/GT(L)/+99C haplotype had lower HO-1 plasma concentrations and lower incidence of multiple organ dysfunction. However, HO-1 polymorphisms were not associated with ICU or hospital mortality. The present study shows that HO-1 is induced in response to stress in both experimental animal models and severely ill patients. HO-1 played an important role in the recovery and repair of infarcted rat hearts. HO-1 induction and CO donor pretreatment enhanced cardiac regeneration after MI, and HO-1 may protect against pathological left ventricular remodeling. Furthermore, HO-1 induction potentially may protect against I/R injury and cardiac dysfunction in isolated rat hearts. In critically ill ICU patients, HO-1 plasma levels correlate with the degree of organ dysfunction, disease severity, and mortality, suggesting that HO-1 may be useful as a marker of disease severity and in the assessment of outcome of critically ill patients.

Myoblast Sheet Transplantation for Treatment of Heart Failure after Myocardial Infarction

Heart failure is a common, severe, and progressive condition associated with high mortality and morbidity. Because of population-aging in the coming decades, heart failure is estimated to reach epidemic proportions. Current medical and surgical treatments have reduced mortality, but the prognosis for patients has remained poor. Transplantation of skeletal myoblasts has raised hope of regenerating the failing heart and compensating for lost cardiac contractile tissue. In the present work, we studied epicardial transplantation of tissue-engineered myoblast sheets for treatment of heart failure. We employed a rat model of myocardial infarction-induced acute and chronic heart failure by left anterior descending coronary artery ligation. We then transplanted myoblast sheets genetically modified to resist cell death after transplantation by expressing antiapoptotic gene bcl2. In addition, we evaluated the regenerative capacity of myoblast sheets expressing the cardioprotective cytokine hepatocyte growth factor in a rat chronic heart failure model. Furthermore, we utilized in vitro cardiomyocyte and endothelial cell culture models as well as microarray gene expression analysis to elucidate molecular mechanisms mediating the therapeutic effects of myoblast sheet transplantation. Our results demonstrate that Bcl2-expression prolonged myoblast sheet survival in rat hearts after transplantation and induced secretion of cardioprotective, proangiogenic cytokines. After acute myocardial infarction, these sheets attenuated left ventricular dysfunction and myocardial damage, and they induced therapeutic angiogenesis. In the chronic heart failure model, inhibition of graft apoptosis by Bcl-2 improved cardiac function, supported survival of cardiomyocytes in the infarcted area, and induced angiogenesis in a vascular endothelial growth factor receptor 1- and 2-dependent mechanism. Hepatocyte growth factor-secreting myoblast sheets further enhanced the angiogenic efficacy of myoblast sheet therapy. Moreover, myoblast-secreted paracrine factors protected cardiomyocytes against oxidative stress in an epidermal growth factor receptor- and c-Met dependent manner. This protection was associated with induction of antioxidative genes and activation of the unfolded protein response. Our results provide evidence that inhibiting myoblast sheet apoptosis can enhance the sheets efficacy for treating heart failure after acute and chronic myocardial infarction. Furthermore, we show that myoblast sheets can serve as vehicles for delivery of growth factors, and induce therapeutic angiogenesis in the chronically ischemic heart. Finally, myoblasts induce, in a paracine manner, a cardiomyocyte-protective response against oxidative stress. Our study elucidates novel mechanisms of myoblast transplantation therapy, and suggests effective means to improve this therapy for the benefit of the heart failure patient.