Adrian Stankiewicz

A new recombinant thrombolytic and antithrombotic agent with higher fibrin affinity – a staphylokinase variant - An in-vivo study

The recombinant protein SAK-RGD-K2-Hir is characterized by its fibrin-specific properties of plasminogen activation combined with antithrombin and antiplatelet activities. It was previously shown in our in-vitro studies to be a more potent and faster-acting thrombolytic agent compared with standard r-SAK. In order to document the effects of the thrombolytic potential of SAK-RGD-K2-Hir we examined this protein in an electrically induced carotid artery thrombosis model and stasis-induced venous model in rats. In the arterial thrombosis model, a bolus injection of SAK-RGD-K2-Hir was less effective than rt-PA and r-SAK. However, the most effective in the improvement and maintenance of carotid patency and in arterial thrombus mass reduction was SAK-RGD-K2. In contrast, all r-SAK derivatives reduced venous thrombus weight significantly in comparison to r-SAK and r-Hir. However, the most observable decrease in thrombus weight was obtained after application of recombinant proteins containing the r-Hir. The bleeding time was significantly prolonged in the animals treated with proteins containing r-Hir at a dose of 1.0 mg/kg. There were no observable changes in plasma fibrinogen concentration. In conclusion, our findings show thrombolytic activity in intravenous bolus injection of the novel thrombolytic agent SAK-RGD-K2-Hir in rats. Although this protein compares favourably with r-SAK in rat venous thrombolysis, we were unable to confirm the beneficial effects of SAK-RGD-K2-Hir over r-SAK and rt-PA in the carotid artery thrombolysis model. Furthermore, our results also suggest that SAK-RGD-K2-Hir bears a risk of bleeding, but this may be true for higher doses.

Study of the mechanisms of aldosterone prothrombotic effect in rats.

Introduction: We investigated the role of primary haemostasis, fibrinolysis, nitric oxide (NO) and oxidative stress as well as mineralocorticoid receptors (MR) in acute aldosterone prothrombotic action. Materials and methods: Venous thrombosis was induced by stasis in Wistar rats. Aldosterone (ALDO; 10, 30, 100 µg/kg/h) was infused for 1 h. Eplerenone (EPL; 100 mg/kg, p.o.), a selective MR antagonist, was administered before ALDO infusion. Bleeding time (BT) and platelet adhesion to collagen were evaluated. The expression of nitric oxide synthase (NOS), NADPH oxidase, superoxide dismutase (SOD) and plasminogen activator inhibitor (PAI-1) was measured. NO, malonyl dialdehyde (MDA) and hydrogen peroxide (H2O2) plasma levels were assayed. Results: Significant enhancement of venous thrombosis was observed after ALDO infusion. ALDO shortened BT and increased platelet adhesion. Marked increases were observed in PAI-1, NADPH oxidase and SOD mRNA levels. MDA and H2O2 levels were augmented in ALDO-treated groups, and NOS expression and NO level were decreased. EPL reduced ALDO effects on thrombus formation, primary haemostasis, PAI-1 expression and MDA level. Conclusion: Short-term ALDO infusion enhances experimental venous thrombosis in the mechanism involving primary haemostasis, fibrinolysis, NO and oxidative stress-dependent pathways. The MR antagonist only partially diminished the ALDO effects, suggesting the involvement of additional mechanisms.

Acute aldosterone infusion enhances thrombosis development in normotensive rats.

Thromb Haemost 2007; 98: 697–699 Dear Sir, It is commonly believed that the morning surge in blood pressure, along with haemostatic changes that promote thrombosis, contributes to cardiovascular events and death at this time of the day. Some studies have shown that aldosterone has circadian rhythmicity and that the major peak of aldosterone occurs during early morning hours (1). It has also been shown that acute systemic infusion of aldosterone in normotensive volunteers results in endothelial dysfunction (2). This observation suggests that even a short-lasting rise in aldosterone level may potentially promote the thrombotic process, by reducing the antithrombotic properties of the endothelium. Therefore, in the present study we wanted to elucidate whether acute infusion of aldosterone could affect experimental thrombosis in rats.The contribution of coagulation and fibrinolytic systems in the mode of aldosterone action was also determined. Furthermore, we investigated the role of the mineralocorticoid receptor in the mechanisms of aldosterone action. Male Wistar rats (300–350 g) were used in this study. All investigations were carried out at the same time of the day (09:00 a.m.) to minimize any effect of diurnal variation in the haemostatic system and vascular function. Aldosterone (ALDO) (Sigma-Aldrich, Poland; 3, 10 and 30 μg/kg/h) or VEH (0.4% ethanol; 2 ml/kg/h) was infused into the femoral vein 5 minutes (min) before the induction of venous thrombosis and was continued for 1 hour (h) and 5 min (Constant-Rate Infusion Pump, Kwapisz, Poland). Eplerenone (EP), a selective mineralocorticoid receptor antagonist (Pfizer Co., Poland), was administered (p.o., 5% aqueous gummi arabici solution) at a dose of 100 mg/ kg 30 min beforeALDO (30 μg/kg/h) infusion. Venous thrombosis was performed by ligation of the vena cava (3), following pentobarbital anesthesia (Vetbutal, Biowet, Poland, 45 mg/kg i.p.). After 1 h, blood samples were drawn from the right ventricle of the heart prior to thrombus removal. The blood was mixed with 3.13% sodium citrate (Sigma-Aldrich, Poland) in a volume ratio of 9:1 and centrifuged for 20 min at 3,500 x g, at 4oC, and then plasma was deep-frozen (-70oC). The venous thrombus was carefully collected from the vein and weighed. Serum aldosterone concentration was measured by the RIA procedure (DPC, Poland). Plasminogen activator inhibitor (PAI-I), tissue plasminogen activator (t-PA), thrombin activatable fibrinolysis inhibitor (TAFI) and tissue factor (TF) plasma levels Letters to the Editor

Ebelactone B, an inhibitor of extracellular cathepsin A-type enzyme, suppresses platelet aggregation ex vivo in renovascular hypertensive rats.

The present study was undertaken to investigate whether ebelactone B, an inhibitor of bradykinin and angiotensin I hydrolysis by serine carboxypeptidase Y-like enzymes, could influence platelet aggregation ex vivo in renovascular hypertensive rats (2-kidney, 1-clip Goldblatt model, 2K1C). We found that ebelactone B (5 mg/kg) administrated subcutaneously once a day for 5 days, 5 weeks after the development of hypertension, or a single dose of ebelactone B (0.5 mg/kg) injected intravenously into 2K1C hypertensive rats before the induction of arterial thrombosis, both markedly suppressed collagen-induced platelet aggregation in whole blood. In contrast, inhibition of collagen-induced platelet aggregation was not evident in vitro after pretreatment of the blood with ebelactone B, indicating that ex vivo the antiaggregatory action of this compound can proceed through an indirect mechanism. The injection of ebelactone B did not affect the mean blood pressure of 2K1C hypertensive rats but lowered an elevated extracellular serine carboxypeptidase cathepsin A-like activity. Thus, the data indicate that ebelactone B may be a promising antiaggregatory agent in renovascular hypertension and suggest that 1 of the possible mechanisms through which it exerts this effect may be related to the suppression of cathepsin A-like activity released locally during the development of renovascular hypertension.

The acute prothrombotic effect of aldosterone in rats is partially mediated via angiotensin II receptor type 1

INTRODUCTION We showed previously that the prothrombotic effect of one hour aldosterone (ALDO) infusion in rats was only partially mediated by the mineralocorticoid receptor (MR). Bearing in mind that ALDO potentiates the effects of angiotensin II (Ang II), in the present study we investigated the role of Ang II receptor type 1 - AT1 in acute ALDO prothrombotic action. MATERIALS AND METHODS The experiments were performed in a stasis-induced venous thrombosis model in male Wistar, normotensive rats. ALDO (30μg/kg) was infused for 1h. Valsartan (VAL; 10mg/kg), a selective AT1 receptor antagonist, was administered in a single bolus injection before ALDO infusion. Eplerenone (EPL, 100mg/kg), a selective MR receptor antagonist, was administered per os before ALDO. Thrombus weight and incidences of thrombosis were assayed. Bleeding time and platelet adhesion to collagen were evaluated as primary hemostasis parameters. The plasma levels of some coagulation and fibrinolysis parameters, and plasma NO metabolite levels were assayed. RESULTS AT1 blockade with valsartan significantly reduced ALDO-induced thrombosis expressed as a reduced thrombus mass (p<0.05 vs ALDO) and diminished the incidence of thrombosis. Valsartan reduced the ALDO-induced changes in bleeding time and platelet adhesion, as well as in coagulation, fibrinolysis, and NO metabolite levels. The effect of AT1 blockade in ALDO-induced thrombosis was similar to the effect of MR blockade. However, dual blockade of AT1 and MR showed no additional benefit. CONCLUSIONS ALDO prothrombotic action is partially mediated via AT1 receptor in the mechanism involving enhanced platelet activation, induced coagulation, impaired fibrinolysis and reduced NO bioavailability.

Thrombocytopenia and perioperative complications after stentless Freedom Solo valve implantation.

BACKGROUND Freedom Solo (FS) stentless bioprostheses have superior haemodynamic performance compared to stented valves; however, the data of thrombocytopenia after FS implantations is disturbing. AIM To compare platelet count and perioperative complications between stentless and stented biological valves in patients undergoing aortic valve replacement. METHODS In 29 patients, FS bovine valves (Sorin Group, Saluggia, Italy) were implanted. Platelet counts were analysed before surgery, on the day of operation, on four consecutive postoperative days (POD) as well as at discharge, and compared to 29 control patients with biological stented porcine valves (Labcor Laboratorios TLBP-A Supra). The analysis of the perioperative variables extracorporeal circulation (ECC), aortic cross clamping (XC) and mechanical ventilation times, as well as blood supply, was performed. RESULTS Initial platelet counts were comparable in both groups. In the FS group, platelet levels on the four consecutive POD were significantly lower. The lowest platelet value (13 × 10³/μL), related to fatal thrombotic thrombocytopenic purpura, was found in one patient from the FS group. ECC as well as XC and mechanical ventilation times, were significantly longer in the FS group, and more blood transfusions in these patients were required. In multiple regression analysis, ECC and XC times did not correlate with platelet count. CONCLUSIONS Implantations of FS stentless bioprostheses are related to significantly lower platelet counts. Severe perioperative complications and their relation to thrombocytopenia need further evaluation.

New derivative of staphylokinase SAK-RGD-K2-Hirul exerts thrombolytic effects in the arterial thrombosis model in rats

SAK-RGD-K2-Hir and SAK-RGD-K2-Hirul are recombinant proteins that are derivatives of r-SAK (recombinant staphylokinase). They are characterized by their fibrin-specific plasminogen activation properties and their antithrombin and antiplatelet activities. The difference between these proteins is the presence of the antithrombotic fragment (hirudin or hirulog) in the C-terminal portion of the r-SAK. The aim of the present study was to examine the thrombolytic potentials of SAK-RGD-K2-Hir and SAK-RGD-K2-Hirul in an electrically induced carotid artery thrombosis model in rats and to compare the potentials to that of r-SAK. We determined that a bolus injection of SAK-RGD-K2-Hirul was more effective than one of r-SAK in the improvement and maintenance of carotid patency and in arterial thrombus weight reduction; however, it had the same potency as SAK-RGD-K2-Hir. The bleeding time, prothrombin time and activated partial thromboplastin time were significantly prolonged in the animals that were treated with either dose (1.5 or 3.0 mg/kg) of SAK-RGD-K2-Hir or SAK-RGD-K2-Hirul, whereas no changes were observed in the plasma fibrinogen concentration or the α2 plasmin inhibitor level. r-SAK alone did not change the bleeding time or coagulation parameters. In conclusion, our findings demonstrate the thrombolytic activity of intravenous bolus injection of the novel thrombolytic agent SAK-RGD-K2-Hirul in rats. Although this protein compares favorably with r-SAK, we were unable to show the presence of any beneficial effects of SAK-RGD-K2-Hirul over those of SAK-RGD-K2-Hir. Furthermore, our results suggest that high doses of SAK-RGD-K2-Hirul bear the risk of bleeding.

Quinapril decreases antifibrinolytic and prooxidative potential of propofol in arterial thrombosis in hypertensive rats.

Angiotensin converting enzyme inhibitors and propofol both exert hypotensive action and may affect hemostasis. We investigated the influence of quinapril and propofol on hemodynamics and hemostasis in renal-hypertensive rats with induced arterial thrombosis. Two-kidney, one clip hypertensive rats were treated with quinapril (3.0 mg/kg for 10 days), and then received propofol infusion (15 mg/kg/h) during ongoing arterial thrombosis. The hemodynamic and hemostatic parameters were assayed. Quinapril exerted a hypotensive effect increasing after propofol infusion. Quinapril showed an antithrombotic effect with the platelet adhesion reduction, fibrinolysis enhancement and oxidative stress reduction. Propofol did not influence thrombosis; however, it inhibited fibrinolysis and showed prooxidative action. The effect of propofol on fibrinolysis and oxidative stress was significantly lower in quinapril-pretreated rats. Mortality was increased among rats treated with both drugs together. Our study demonstrates that pretreatment with quinapril reduced the adverse effects of propofol on hemostasis. Unfortunately, co-administration of both drugs potentiated hypotension in rats, which corresponds to higher mortality.

Single-centre experience in surgery of acute aortic type A dissection and true aortic arch aneurysm.

BACKGROUND Surgery of the aortic arch is challenging. AIM To assess the results of aortic arch surgery. METHODS Analysis of 172 patients operated on arch dissection (emergency group: 97 patients) or aneurysm (elective group: 75 patients) between 2007 and 2014. Arch surgery was defined as a procedure requiring circumferential anastomosis at the level of the aortic arch or the descending aorta with the use of techniques of brain protection (deep hypothermic circulatory arrest [DHCA] or selective antegrade cerebral perfusion [SACP]) and/or debranching of at least one supra-aortic vessel. RESULTS Men predominated in both groups (> 70%). Men were younger in the emergency group (55 vs. 66 years; p < 0.008). The operative risk was higher in the emergency group (19.2% vs. 12.5%; p < 0.001). Forty-nine per cent of the patients from the emergency group and 5% from the elective group were operated with antiplatelet therapy (p < 0.001). Extended hemiarch procedure was performed in 79% (n = 77) in the emergency and 76% (n = 57) in the elective group. Total arch replacement was performed in 19 (21%) patients from the emergency and 18 (24%) patients from the elective group. In these patients debranching was performed in 68% of the emergency patients group and in 67% of the elective group. Elephant trunk procedure (classic/frozen) was performed in 53% (n = 10) from the emergency and in 78% (n = 14) of patients from the elective group. Aortic valve sparing surgery was performed in 20% of patients from the emergency and 9% from the elective group (p = 0.063). DHCA was performed in 58% (n = 43) of patients from the elective group and 39% (n = 37) from the emergency group. SACP was performed in 61% (n = 58) of patients from the emergency and 42% (n = 31) from the elective group. Thirty-day mortality in the emergency group reached 33% (n = 32), and in the elective group 15% (n = 11; p = 0.007). In multivariate analysis, predictors of death in the emergency group were: Logistic EuroSCORE above 19.5%, extracorporeal circulation time above 228 min, and postoperative acute renal failure (ARF); and in the elective group: DHCA time above 26 min, rethoracotomy due to bleeding, and ARF. Follow-up was completed in 100% of patients in terms of vital status. The mean follow-up time of the patients from the emergency group was 24.3 ± 27.10 (min 0, max 92) months, and from the elective group 30.3 ± 24.5 (min 0, max 99) months. During the follow-up period all-cause mortality in the emergency group was 43% (n = 42/97), and in the elective group it was 36% (n = 27/75). CONCLUSIONS Early mortality in the emergency group was higher, while long-term mortality did not differ among the groups. Postoperative ARF is a critical predictor of mortality in both groups.

Hybrid thoracoabdominal procedure for acute aortic dissection Stanford type A using the EVITA OPEN PLUS – case report

Rozwarstwienie aorty typu A wg Stanford to stan zagrożenia życia wymagający pilnej interwencji kardiochirurgicznej. Obecnie obok klasycznych metod operacyjnych możliwe jest zastosowanie technik endowaskularnych. Przedmiotem niniejszego artykułu jest prezentacja przypadku 58-letniej chorej leczonej wieloetapowo z powodu rozwarstwienia aorty typu A wg Stanford, z wykorzystaniem technik hybrydowych, u której zakres leczenia objął całą aortę od zastawki aortalnej, z jej wymianą, do tętnic biodrowych. Słowa kluczowe: rozwarstwienie aorty typu A, operacja hybrydowa. Abstract