Novel mutations in the tyrosine hydroxylase gene in the first Czech patient with tyrosine hydroxylase deficiency.

Tyrosine hydroxylase deficiency manifests mainly in early childhood and includes two clinical phenotypes: an infantile progressive hypokinetic-rigid syndrome with dystonia (type A) and a neonatal complex encephalopathy (type B). The biochemical diagnostics is exclusively based on the quantitative determination of the neurotransmitters or their metabolites in cerebrospinal fluid (CSF). The implementation of neurotransmitter analysis in clinical praxis is necessary for early diagnosis and adequate treatment. Neurotransmitter metabolites in CSF were analyzed in 82 children (at the age 1 month to 17 years) with clinical suspicion for neurometabolic disorders using high performance liquid chromatography (HPLC) with electrochemical detection. The CSF level of homovanillic acid (HVA) was markedly decreased in three children (64, 79 and 94 nmol/l) in comparison to age related controls (lower limit 218-450 nmol/l). Neurological findings including severe psychomotor retardation, quadruspasticity and microcephaly accompanied with marked dystonia, excessive sweating in the first patient was compatible with the diagnosis of tyrosine hydroxylase (TH) deficiency (type B) and subsequent molecular analysis revealed two novel heterozygous mutations c.636A>C and c.1124G>C in the TH gene. The treatment with L-DOPA/carbidopa resulted in the improvement of dystonia. Magnetic resonance imaging studies in two other patients with microcephaly revealed postischaemic brain damage, therefore secondary HVA deficit was considered in these children. Diagnostic work-up in patients with neurometabolic disorders should include analysis of neurotransmitter metabolites in CSF.

EkoSonicSV endovascular system for recanalization of the basilar artery occlusion.

The interventional management of stroke may consist of the use of angioplasty, stenting or mechanical thrombus removal technique. For this purpose several retrieval devices are being used. Recently the new alternative device - EkoSonicSV has been introduced, which is particularly suitable for recanalization of the occluded basilar artery (BA). Here we are presenting a complete recanalization of BA using this device in two patients with stroke over a short period of time together with the intra-arterial use of recombinant tissue plasminogen activator and application of intravascular ultrasound.

Endovascular treatment of mycotic pseudoaneurysms.

The surgical correction of ruptured intracranial infectious pseudoaneurysms is associated with high morbidity and mortality. An endovascular therapeutic approach has been introduced recently. This treatment is, compared to surgical intervention, less invasive, faster, more effective and safer, thus making it a gentler option, particularly for pediatric patients. Lower morbidity and mortality have been achieved thanks to the combination of prolonged administration of antibiotics, coil embolization, and parent artery occlusion. Two pediatric cases of bleeding mycotic pseudoaneurysm treated successfully with fibered coil embolization and long-term antibiotics are dealt with in this manuscript.

The potential of RFID technology in Blood Center processes.

Current RFID technology deployment is limited by safety, procedural and physical limitations in healthcare field. It is important to define and ensure safe operation of technologies without actual deployment in real operation. Potential problems could arise due to the consequences of technical and physical characteristics of RFID technology and its improper location. This article deals with manipulation of blood products and the definition of suitable places for radio identification. Each suitable place must undergo laboratory experiments and tests. The results can provide a convenient base for defining efficient and safe deployment of RFID technology in Blood Centers with substantial financial savings for Czech healthcare.

[Prevention of venous thromboembolism in surgery, laparoscopic surgery and urology]. / Prevence zilního tromboembolizmu v chirurgii, laparoskopické chirurgii, urologii.

Deep venous thrombosis and pulmonary embolism are major health problems with potential serious outcomes. Acutely, pulmonary embolism may be fatal. In the long term, pulmonary hypertension can develop from recurrent pulmonary embolism. Often overlooked is post-thrombotic chronic venous insufficiency occurring as a result of deep venous thrombosis causing deep venous reflux or obstruction with skin changes and ulceration with adverse impact on quality of life and escalation of health care costs. Almost all hospitalized patients have at least one risk factor for venous thrombosis and approximately 40% have three or more risk factors. Without thromboprophylaxis, the incidence of objectively confirmed, hospital-acquired deep venous thrombosis is approximately 10 to 40% among medical or general surgical patients and 40 to 60% following major orthopedic surgery. Abundant data from metaanalysis and blinded, randomized clinical trials have demonstrated strong evidence that primary thromboprophylaxis reduces deep venous thrombosis and pulmonary embolism and little or no increase in the rates of clinically important bleeding with prophylactic doses of low-dose unfractionated heparin, low-molecular-weight heparin or fondaparinuxem.

Autologous bone marrow stem cell transplantation in patients with end-stage chronical critical limb ischemia and diabetic foot.

A total 37 patients suffering from end stage-IV Fontaine (CLI and diabetic foot) with an ulcerated limb in whom all previous therapeutic strategies failed (e.g. surgical revascularization and endovascular repair) were selected and underwent local transplantation of Autologous Bone Marrow Stem Cells (ABMSCs). The efficacy/safety ofthis therapy was assessed by using several endpoints such as (a) prevention of amputation, (b) wound healing and (c) degree of angiogenesis. In order to assess the limb ischemia and hypoxia the several tests and measurements were performed pre- and post transplantation at a variety of time intervals. The measurements include: TP-toe pressure measurements (by Periflux 5000 Laser Doppler and Oxymetry system), SPP-skin perfusion pressure, ABI-ankle brachial index, LDP-Laser Doppler baseline and heat perfusion assessment, TcpO2 without and with O2 provocation inhalation test. In addition, a battery of biochemical and hematological tests of peripheral venous blood samples and bone marrow analysis were performed. Limb salvage was 81% in 30 patients, 7 patients (19%) were amputated for terminal severe ischemia and gangrene progression. In the group of limb salvage patients initial Toe pressure 23.119 (std. error 5.358) increased in 90 days follow-up into 29.888 (std. error 5.99), Toe brachial index increased from 0.1469 (std. 0.0326) to 0.1991(std. 0.401). In LASER doppler and TcpO2, TcpCO2 tissue perfusion examination TcpO2% Increase after O2 provocation inhalation test was elevated from 162.95 (%) to 229.86% which confirmed a very good tissue vasoreactivity after BMSC transplantation.

[Bleeding complications of anticoagulant therapy]. / Krvácivé komplikace antikoagulacní lécby.

Anticoagulant therapy is one of the most common forms of medical intervention. It is the mainstay of prevention and treatment of thrombotic events. Omission of adequate anticoagulant prophylaxis at least for moderate-risk and high-risk patients is a widely recognized medical error. Bleeding is one of the most feared complications of anticoagulant therapy, and is a risk of all anticoagulants. Whereas unfractionated heparin and warfarin, the oldest and most widely used anticoagulants, have specific antidotes for their anticoagulant effect, many of the newer agents (direct and indirect inhibitors of coagulation factors Xa and/or IIa) do not have specific antidotes to reverse their actions. The use of novel anticoagulants is further complicated by a lack of easily available laboratory tests to measure their levels and thereby optimize their benefit and safety in clinical practice. In this review, we evaluate the risk of bleeding associated with current anticoagulants, review the data available on current and experimental agents used for the reversal of anticoagulation, and provide recommendations for the management of major bleeding associated with anticoagulant therapy and for the management of asymptomatic overdosing of the anticoagulants.

Alpha 2-antiplasmin supplementation inhibits tissue plasminogen activator-induced fibrinogenolysis and bleeding with little effect on thrombolysis.

Tissue plasminogen activator (t-PA) causes fibrinogen proteolysis when alpha 2-antiplasmin levels fall, and this may contribute to t-PA-induced hemorrhage. Because clot-bound plasmin is protected from alpha 2-antiplasmin inhibition, we tested the possibility that alpha 2-antiplasmin supplementation would block t-PA-induced fibrinogenolysis and bleeding without affecting thrombolysis. When added to human or rabbit plasma, alpha 2-antiplasmin inhibits t-PA-induced fibrinogenolysis, but hat little effect on the lysis of 125I-fibrin clots. To examine its effect in vivo, rabbits with preformed 125I-labeled-jugular vein thrombi were randomized to receive t-PA, t-PA and alpha 2-antiplasmin, or saline. alpha 2-Antiplasmin infusion produced a modest decrease in t-PA-induced thrombolysis (from 40.2% to 30.1%, P = 0.12), but reduced fibrinogen consumption from 87% to 27% (P = 0.0001), and decreased blood loss from standardized ear incisions from 5,594 to 656 microliter (P < 0.0001). We hypothesize that alpha 2-antiplasmin limits t-PA-induced hemorrhage by inhibiting fibrinogenolysis and subsequent fragment X formation because (a) SDS-PAGE and immunoblot analysis indicate less fragment X formation in alpha 2-antiplasmin treated animals, and (b) when added to a solution of fibrinogen and plasminogen clotted with thrombin in the presence of t-PA, fragment X shortens the lysis time in a concentration-dependent fashion. These findings suggest that fragment X incorporation into hemostatic plugs contributes to t-PA-induced bleeding. By blocking t-PA-mediated fibrinogenolysis, alpha 2-antiplasmin supplementation may improve the safety of fibrin-specific plasminogen activators.

Emerging anticoagulants: mechanism of action and future potential.

Medical needs associated with diverse thromboembolic conditions are not fully met by currently available anticoagulants. Of those, unfractionated heparin (UFH) is gradually replaced by low molecular weight heparin (LMWH) for prevention and treatment of venous thromboembolism and acute coronary syndromes, along with supportive treatment with oral anticoagulants, such as warfarin derivatives. While generally effective these agents have several shortcomings involving compliance, delivery, efficacy and safety considerations in various disease settings, and for these reasons new anticoagulants are sought, to target more specifically the critical effectors and steps in the blood coagulation process, namely: (i) initiation, (ii) propagation and (iii) the phase of thrombin activity. The emerging agents that block tissue factor/factor VIIa-dependent initiation phase of the coagulation cascade, include: recombinant tissue factor pathway inhibitor (rTFPI), nematode anticoagulant peptide (NAPc2), active site-blocked factor VIIa (FVIIai) and TF targeting antibodies. Some of them are currently evaluated in clinical trials with promising results. Propagation phase of thrombus formation (e.g. the activity of factors IXa, Xa, VIIIa or Va) is targeted mainly by various indirect, direct and bimodal inhibitors, such as fondaparinux, indraparinux, tick anticoagulant peptide (TAP), antistatin (ANT) and antithrombin-heparin covalent complex (ATH), all endowed mostly with an anti-Xa activity. Although promising, some of these agents (TAP, ANT and ATH) have not progressed beyond animal testing while others (fondaparinux) was already assessed for prevention and treatment of venous thromboembolism and for treatment of arterial thrombosis. Lastly, inhibitors of thrombin activity are composed of either indirect (UFH, LMWH), or direct thrombin (FIIa) inhibitors including: hirudin, argatroban, melagatran, ximelagatran, dabigatran, and bivalirudin. These agents are either in advanced development or already approved for clinical use. Bimodal FIIa inhibitory activity of ATH was demonstrated in animal models of venous and arterial thrombosis, but is in need of further development. In conclusion, while some of these emerging anticoagulants, such as fondaparinux, idraparinux, ximelagatran and ATH appear to possess superior efficacy-safety profile, as compared to their conventional predecessors (UFH, LMWH and warfarin), their cost-effectiveness, side effects and antidote availability have to be considered. More importantly, coagulation factors that are targets of these inhibitory activities also affect coagulation independent processes, such as wound healing, inflammation, angiogenesis, mitogenesis and cell survival. Thus the consequences of both coagulation-dependent and -independent effects of new agents should be carefully considered before proper clinical indications are established.

Genetic determinants of cancer coagulopathy, angiogenesis and disease progression.

Progression of human malignancies is accompanied by vascular events, such as formation and remodeling of blood vessels and systemic coagulopathy. Though long appreciated as comorbidity of cancer (Trousseau syndrome), vascular involvement is increasingly recognized as a central pathogenetic mechanism of tumor growth, invasion and metastasis. The major outstanding question in relation to this role has been, whether vascular perturbations are simply a reaction to the conditions of the tumor microenvironment, or are linked to the known genetic lesions causal for the onset and progression of malignancy. In this regard, we have previously hypothesized, and recently demonstrated experimentally that deregulation of certain hemostatic mechanisms, namely upregulation of tissue factor (TF) and possibly other changes (e.g. expression of thrombin receptor - PAR-1) are controlled by cancer-associated oncogenic events, such as activation of K-ras, epidermal growth factor receptor (EGFR), or inactivation of the p53 tumor suppressor gene in various human cancer cells. It appears that these respective transforming alterations exert their impact on both, cell-associated and soluble/circulating (microvesicle- associated) TF, i.e. may cause a systemic hypercoagulable state. Other genes, which more recently emerged as regulators of cancer coagulopathy include: PML-RARalpha, PTEN, and MET. While the spectrum of procoagulant targets of these genes may vary somewhat it includes: TF, PAI-1, COX-2 and possibly other hemostatic proteins. It is noteworthy that these prothrombotic changes may impact the malignant process directly (e.g. stimulate angiogenesis, tumor growth or metastasis) as a consequence of both coagulation-dependent and -independent effects. The latter are mostly related to cellular signaling events and changes in gene expression which are now known to be induced by the TF/FVIIa/Xa complex, thrombin and PARs, expressed on the surface of cancer cells, as well as tumor-associated endothelium. Interestingly, certain anticoagulants possess antimetastatic and anticancer properties (e.g. LMWH), an observation that further suggests that hypercoagulability may act as an effector mechanism of genetically driven tumor progression. Conversely, we suggest that oncogene-directed (targeted) anticancer agents could, at least in some cases, ameliorate not only cellular transformation itself, but also some of the chronic components of the cancer-related coagulopathy, something that may be relevant to therapeutic efficacy of these drugs. We also postulate that since TF is the oncogene target, circulating TF (microparticles) could serve as surrogate marker of the biological activity oncogene-directed agents exert in vivo. Thus, both genetic and epigenetic factors appear to conspire to activate various components of the hemostatic system in cancer patients, both locally and systemically. These activities act as mediators of cancer coagulopathy, angiogenesis, metastasis and other events involved in disease progression and should be recognized in designing better anticancer therapies.

Assembly of mitochondrial ATP synthase in cultured human cells: implications for mitochondrial diseases.

To study the assembly of mitochondrial F1F0 ATP synthase, cultured human cells were labeled with [35S]methionine in pulse-chase experiments. Next, two-dimensional electrophoresis and fluorography were used to analyze the assembly pattern. Two assembly intermediates could be demonstrated. First the F1 part appeared to be assembled, and next an intermediate product that contained F1 and subunit c. This product probably also contained subunits b, F6 and OSCP, but not the mitochondrially encoded subunits a and A6L. Both intermediate complexes accumulated when mitochondrial protein synthesis was inhibited, suggesting that mitochondrially encoded subunits are indispensable for the formation of a fully assembled ATP synthase complex, but not for the formation of the intermediate complexes. The results and methods described in this study offer an approach to study the effects of mutations in subunits of mitochondrial ATP synthase on the assembly of this complex. This might be of value for a better understanding of deficiencies of ATP synthase activity in mitochrondrial diseases.

Differentiation of brown adipose tissue and biogenesis of thermogenic mitochondria in situ and in cell culture.

Differentiation and biogenesis of mitochondria in brown adipose tissue (BAT) was studied in situ and in cell culture by Western blotting, enzyme activity measurements, [35S]methionine incorporation and immunofluorescence microscopy. In different rodent species the perinatal development of BAT thermogenic function resulted from the formation of thermogenic mitochondria which replaced the preexisting nonthermogenic mitochondria. Their biogenesis was characterized by the sudden appearance and rapid increase of the uncoupling protein (UCP), increase of cytochrome oxidase (COX) and decrease of H(+)-ATPase. In primary cell culture, differentiation of precursor cells from mouse BAT to typical multilocular adipocytes was accompanied by increasing content of COX and H(+)-ATPase. A selective synthesis of UCP was induced by activation of beta-adrenergic receptors or by elevated levels of cellular cAMP. UCP was quantitatively incorporated into mitochondria and within 24 h after stimulation reached near physiological concentration. Both in situ and in cell culture, the conditions enabling the expression of UCP gene were accompanied by activation of intracellular thyroxine 5'-deiodinase.

Altered properties of mitochondrial ATP-synthase in patients with a T-->G mutation in the ATPase 6 (subunit a) gene at position 8993 of mtDNA.

A family is described with a T-->G mutation at position 8993 of mtDNA. This mutation is located in the ATPase 6 gene of mtDNA which encodes subunit a of the ATP-synthase complex (FlFo-ATPase). Clinically, the patients showed severe infantile lactate acidosis and encephalomyopathy in a form that was different from the classical Leigh syndrome. In 3 affected boys, ranging in age from 3 months to 8 years, the mutation was found in 95-99% of the mtDNA population. The clinical symptoms correlated with the mtDNA heteroplasmy and in the healthy mother 50% of the mtDNA was mutated. The rate of mitochondrial ATP production by cultured skin fibroblasts containing 99% of mutated mtDNA was about 2-fold lower than that in normal fibroblasts. Native electrophoresis of the mitochondrial enzyme complexes revealed instability of the FlFo-ATPase in all the tissues of the patient that were investigated (heart, muscle, kidney, liver). Only a small portion of the ATP-synthase complex was present in the complete, intact form (620 kDa). Incomplete forms of the enzyme were present as subcomplexes with approx. molecular weights of 460, 390 and 150 kDa, respectively, which differed in the content of F1 and Fo subunits. Immunochemical analysis of the subunits of the FlFo-ATPase further revealed a markedly decreased content of the Fo subunit b in mitochondria from muscle and heart, and an increased content of the Fo subunit c in muscle mitochondria, respectively. These results indicate that in this family the T-->G point mutation at position 8993 in the mitochondrial ATPase 6 gene is accompanied by structural instability and altered assembly of the enzyme complex, that are both most likely due to changes in the properties of subunit a of the membrane sector part of the ATP-synthase.

Expression and fate of the nuclearly encoded subunits of cytochrome-c oxidase in cultured human cells depleted of mitochondrial gene products.

Synthesis, import, assembly and turnover of the nuclearly encoded subunits of cytochrome-c oxidase were investigated in cultured human cells depleted of mitochondrial gene products by continuous inhibition of mitochondrial protein synthesis (OP- cells). Immunoprecipitation after pulse labeling demonstrated that the synthesis of the nuclear subunits was not preferentially inhibited, implying that there is no tight regulation in the synthesis of mitochondrial and nuclear subunits of mitochondrial enzyme complexes. Quantitative analysis of the mitochondrial membrane potential in OP- cells indicated that its magnitude was about 30% of that in control cells. This explains the normal import of the nuclearly encoded subunits of cytochrome-c oxidase and other nuclearly encoded mitochondrial proteins into the mitochondria that was found in OP- cells. The turnover rate of nuclear subunits of cytochrome-c oxidase, determined in pulse-chase experiments, showed a specific increase in OP- cells. Moreover, immunoblotting demonstrated that the steady-state levels of nuclear subunits of cytochrome-c oxidase were severely reduced in these cells, in contrast to those of the F1 part of complex V. Native electrophoresis of mitochondrial enzyme complexes showed that assembly of the nuclear subunits of cytochrome-c oxidase did not occur in OP- cells, whereas the (nuclear) subunits of F1 were assembled. The increased turnover of the nuclear subunits of cytochrome-c oxidase in OP- cells is, therefore, most likely due to an increased susceptibility of unassembled subunits to intra-mitochondrial degradation.

Altered kinetics of cytochrome c oxidase in a patient with severe mitochondrial encephalomyopathy.

Deficiency of cytochrome c oxidase activity was established in a girl born to consanguineous parents. She showed symptoms of dysmaturity, generalized hypotonia, myoclonic seizures and progressive respiratory failure, leading to death on the seventh day of life. Structural abnormalities of the central nervous system consisted of severe cerebellar hypoplasia and optic nerve atrophy. Biochemical analysis of a muscle biopsy specimen demonstrated deficiency of cytochrome c oxidase activity. Cultured fibroblasts from this patient also showed a selective decrease in the activity of cytochrome c oxidase, excluding a muscle-specific type of deficiency. Further investigations in cultured fibroblasts revealed that synthesis, assembly and stability of both the mitochondrial and the nuclear subunits of the enzyme were entirely normal. The steady-state concentration of cytochrome c oxidase in the fibroblasts of the patient was also normal, suggesting that the kinetic properties of the enzyme were altered. Analysis of the kinetic parameters of cytochrome c oxidase demonstrated an aberrant interaction between cytochrome c oxidase and its substrate, cytochrome c, most likely because of a mutation in one of the nuclear subunits of the enzyme.

Heparin compromises streptokinase-induced arterial patency in rabbits.

INTRODUCTION: Little is known with regard to efficacy of heparin as an adjunct to fibrinolytics under conditions of severe vascular damage. In this study, we compared the effects of unfractionated heparin (UH), low-molecular weight heparin (LMWH), and recombinant desulfohirudin (HIR) in combination with streptokinase (SK) in such settings. MATERIALS AND METHODS: We used an established rabbit model, in which thrombosis, critical stenosis, and vascular wall damage were introduced to a segment of the abdominal aorta and the effects of the respective therapies were assessed by time to patency (TTP in minutes), cumulative patency (CP (%)), lysis of original clot (CL (%)), and net clot accretion (NCA (%)). Treatments were administered over 90 min at the following doses: SK: 33,000 U/kg, UH: 125-250 U/kg, LMWH: 1.25-2.5 mg/kg, HIR: 0.25-0.55 mg/kg. RESULTS: Unexpectedly, UH and LMWH had a paradoxical and detrimental effect on SK-mediated recanalization as measured by both TTP and CP. Thus, administration of SK vs. SK+UH or SK+LMWH resulted in TTP values of 43+/-8 min vs. 70+/-11 min (p<0.05) and 67+/-12 min. (p<0.08), respectively. For CP, the corresponding values were 21+/-7%, 0.5+/-0.3% and 9+/-8%. This delay in vessel recanalization occurred despite excessive systemic anticoagulation (activated partial thromboplastin time (aPTT) and thrombin clotting time (TCT) ratios >6 and >34, respectively). Of interest, both heparinoids completely inhibited SK-induced fibrinogen consumption (FC). In contrast, recombinant desulfohirudin (HIR) shortened SK-induced TTP (4.97+/-0.81 min) without preserving fibrinogen. CONCLUSIONS: Our findings suggest that caution needs to be exercised, when using the combination of SK and heparinoids for the treatment of arterial thrombosis under conditions of severe vascular damage and stenosis.

The hemostatic system and angiogenesis in malignancy.

Coagulopathy and angiogenesis are among the most consistent host responses associated with cancer. These two respective processes, hitherto viewed as distinct, may in fact be functionally inseparable as blood coagulation and fibrinolysis, in their own right, influence tumor angiogenesis and thereby contribute to malignant growth. In addition, tumor angiogenesis appears to be controlled through both standard and non-standard functions of such elements of the hemostatic system as tissue factor, thrombin, fibrin, plasminogen activators, plasminogen, and platelets. "Cryptic" domains can be released from hemostatic proteins through proteolytic cleavage, and act systemically as angiogenesis inhibitors (e.g., angiostatin, antiangiogenic antithrombin III aaATIII). Various components of the hemostatic system either promote or inhibit angiogenesis and likely act by changing the net angiogenic balance. However, their complex influences are far from being fully understood. Targeted pharmacological and/or genetic inhibition of pro-angiogenic activities of the hemostatic system and exploitation of endogenous angiogenesis inhibitors of the angiostatin and aaATIII variety are under study as prospective anti-cancer treatments.

The benefit-to-risk profile of melagatran is superior to that of hirudin in a rabbit arterial thrombosis prevention and bleeding model.

Although hirudin is better than heparin at preventing recurrent ischemia in patients with unstable angina, hirudin produces more bleeding. The purpose of this study was to use a rabbit arterial thrombosis prevention and ear bleeding model to determine whether for equivalent efficacy, melagatran, a synthetic direct thrombin inhibitor, is safer than hirudin. A combination of balloon injury and stasis was used to induce thrombosis in the distal aorta, and patency and blood flow were continuously monitored with ultrasonic flow probes. Rabbits were randomized to melagatran (in total doses of 78-313 nmol kg(-1)), hirudin (in total doses of 18-107 nmol kg(-1)), or saline over 90 min. To assess safety, blood loss from standardized ear incisions was measured. Both melagatran and hirudin produced dose-dependent increases in patency and blood flow. At doses that maintained the highest levels of patency, however, melagatran produced 2-3-fold less bleeding than hirudin. Thus, at maximally effective doses, melagatran causes less bleeding than hirudin in this model. These findings raise the possibility that some direct thrombin inhibitors are safer than others.

The effect of thrombin inhibitors on tissue plasminogen activator induced thrombolysis in a rat model.

Successful coronary thrombolysis depends on rapidly restoring blood flow and maintaining patency of the infarct-related artery. Although widely used as an adjunct to lytic therapy, heparin is limited in its ability to produce these effects. Since the limitations of heparin may reflect its inability to inactivate clot-bound thrombin, we developed a rat model of tissue plasminogen activator (t-PA) induced thrombolysis to compare doses of heparin, hirudin, hirulog (a synthetic hirudin-derived peptide), and D-Phe-Pro-ArgCH2Cl (PPACK) that produced a 4-fold prolongation of the baseline activated partial thromboplastin time (APTT) with saline in terms of their ability to accelerate thrombolysis and to prevent reocclusion. A thrombus rich in red cells and fibrin was formed in the distal aorta by applying an external constrictor after denuding the endothelium with a balloon catheter. Thrombolysis was induced with t-PA (1 mg/kg bolus, followed by 1 mg kg-1 h-1 over 30 min) and the rats were then randomized to receive a concomitant 80 min infusion of a thrombin inhibitor or saline. By continuously monitoring blood flow and pre- and post-stenotic blood pressures, the time to clot lysis, and the number of reocclusions were determined. Compared to saline, heparin had no significant effect on these variables.(ABSTRACT TRUNCATED AT 250 WORDS)

Multidose blood versus crystalloid cardioplegia. Comparison by quantitative assessment of irreversible myocardial injury.

The relative efficacy and safety of blood-based potassium cardioplegic solutions compared to crystalloid arresting solutions has been a major controversy in the field of intraoperative myocardial protection for cardiac operations. In this study multidose potassium (K+ = 30 mEq/L) blood cardioplegia was compared to multidose potassium crystalloid cardioplegia in a dog model in which hearts were arrested for periods of 4 1/2 and 6 hours. The cardioplegic solution was given as an initial bolus of 500 ml and then as 250 ml doses every 30 minutes of arrest. In the 4 1/2 hour arrest group, six animals received blood cardioplegia, six received a low-sodium crystalloid cardioplegia (modified Roe's solution), and 10 received a high sodium crystalloid cardioplegic solution of our own design. In the 6 hour arrest group, four animals received blood cardioplegia, four received the low-sodium solution, and four received the high-sodium solution. Myocardial temperature was precisely controlled at 27 degrees +/- 1 degree C in all groups. The hearts were reperfused for periods of 2 to 4 hours after the arrest periods and then examined morphologically for injury. The extent of myocardial damage was quantified in 5 mm thick transverse sections through the ventricles by using a tetrazolium enzyme-mapping technique. In the crystalloid groups the hearts arrested for 4 1/2 hours were significantly injured. The percentage (+/- SEM) of necrosis was 12.3 % +/- 5.6% in the low-sodium cardioplegic (modified Roe's) group and 9.3% +/- 3.4% in the high-sodium group. In the 6 hour arrest group the hearts were severely injured, with contracture occurring in all cases. The percentage of necrosis was 56.5% +/- 13% in the low-sodium cardioplegic group and 71.3% +/- 12% in the high-sodium group. In striking contrast all hearts protected with blood cardioplegia failed to show any evidence of tissue damage either on tetrazolium staining or on electron microscopic examination. We conclude that blood cardioplegia offers superior protection to the arrested heart at moderate hypothermia compared to crystalloid cardioplegia.