Transgenic Expression of Human CD46 on Porcine Endothelium: Effect on Coagulation and Fibrinolytic Cascades During Ex Vivo Human-to-Pig Limb Xenoperfusions

BACKGROUND Dysregulation of the coagulation system due to inflammatory responses and cross-species molecular incompatibilities represents a major obstacle to successful xenotransplantation. We hypothesized that complement inhibition mediated by transgenic expression of human CD46 in pigs might also regulate the coagulation and fibrinolysis cascades and tested this in ex vivo human-to-pig xenoperfusions. METHODS Forelimbs of wild-type and hCD46/HLA-E double transgenic pigs were ex vivo xenoperfused for 12 hours with whole heparinized human blood. Muscle biopsies were stained for galactose-α1,3-galactose, immunoglobulin M, immunoglobulin G, complement, fibrin, tissue factor, fibrinogen-like protein 2, tissue plasminogen activator (tPA), and plasminogen activator inhibitor (PAI)-1. The PAI-1/tPA complexes, D-dimers, and prothrombin fragment F1 + 2 were measured in plasma samples after ex vivo xenoperfusion. RESULTS No differences of galactose expression or deposition of immunoglobulin M and immunoglobulin G were found in xenoperfused tissues of wild type and transgenic limbs. In contrast, significantly lower deposition of C5b-9 (P < 0.0001), fibrin (P = 0.009), and diminished expression of tissue factor (P = 0.005) and fibrinogen-like protein 2 (P = 0.028) were found in xenoperfused tissues of transgenic limbs. Levels of prothrombin fragment F1 + 2 (P = 0.031) and D-dimers (P = 0.044) were significantly lower in plasma samples obtained from transgenic as compared to wild-type pig limb perfusions. The expression of the fibrinolytic marker tPA was significantly higher (P = 0.009), whereas PAI-1 expression (P = 0.022) and PAI-1/tPA complexes in plasma (P = 0.015) were lower after transgenic xenoperfusion as compared to wild-type xenoperfusions. CONCLUSIONS In this human-to-pig xenoperfusion model, complement inhibition by transgenic hCD46 expression led to a significant inhibition of procoagulant and antifibrinolytic pathways.

Endovascular Treatment of Acute Stroke: Evolution and Selection of Techniques and Instruments Based on Thrombus Imaging.

Mechanical thrombectomy provides higher recanalization rates than intravenous or intra-arterial thrombolysis. Finally this has been shown to translate into improved clinical outcome in six multicentric randomized controlled trials. However, within cohorts the clinical outcomes may vary, depending on the endovascular techniques applied. Systems aiming mainly for thrombus fragmentation and lacking a protection against distal embolization have shown disappointing results when compared to recent stent-retriever studies or even to historical data on local arterial fibrinolysis. Procedure-related embolic events are usually graded as adverse events in interventional neuroradiology. In stroke, however, the clinical consequences of secondary emboli have so far mostly been neglected and attributed to progression of the stroke itself. We summarize the evolution of instruments and techniques for endovascular, image-guided, microneurosurgical recanalization in acute stroke, and discuss how to avoid procedure-related embolic complications.

Acute mental stress and hemostasis: When physiology becomes vascular harm

Stress-induced activation of the sympathoadrenal medullary system activates both the coagulation and fibrinolysis system resulting in net hypercoagulability. The evolutionary interpretation of this physiology is that stress-hypercoagulability protects a healthy organism from excess bleeding should injury occur in fight-or-flight situations. In turn, acute mental stress, negative emotions and psychological trauma also are triggering factors of atherothrombotic events and possibly of venous thromboembolism. Individuals with pre-existent atherosclerosis and impaired endothelial anticoagulant function are the most vulnerable to experience onset of acute coronary events within two hours of intense emotions. A range of sociodemographic and psychosocial factors (e.g., chronic stress and negative affect) might critically intensify and prolong stress-induced hypercoagulability. In contrast, several pharmacological compounds, dietary flavanoids, and positive affect mitigate the acute prothrombotic stress response. Studies are needed to investigate whether attenuation of stress-hypercoagulability through medications and biobehavioral interventions reduce the risk of thrombotic incidents in at-risk populations.

Factores trombogénicos sistémicos en paciente con síndrome coronario agudo con cinecoronariografía normal y patológica

En la fisiopatología del síndrome coronario agudo (SCA) sin evidencia de lesión ateromatosa oclusiva, podría participar un estado de hipertrombogenicidad sanguínea, generado por factores trombogénicos sistémicos, como los factores de riesgo cardiovascular y los que intervienen en el balance coagulación-anticoagulación, fibrinolisis y formación de fibrina. Objetivo: Estudiar y comparar los factores trombogénicos sistémicos en pacientes con SCA y cinecoronariografía (CCG) normal y patológica.

Migration of keratinocytes through tunnels of digested fibrin

We report here a hitherto undescribed form of cell migration. When a suspension of human keratinocytes is plated on a fibrin matrix, single cells invade the matrix and progress through it as rounded cells by dissolving the fibrin and thereby creating tunnels. These tunnels are cylindrical or helical, the latter being the result of constant change in the path of cellular advance around the helical axis. Helical tunnel formation is strongly promoted by epidermal growth factor. The rate of migration of the cell through the track of a helical tunnel (up to 2.1 mm per day) is about 7-fold greater than through a cylindrical tunnel. Pericellular fibrinolysis leading to tunnel formation depends on the presence of plasminogen in the medium and its conversion to plasmin by a cellular activator. Formation of tunnels requires that plasminogen activator be localized on the advancing surface of the keratinocyte; we propose that the tunnel is cylindrical when the site of release of plasmin is located at a fixed point on the cell surface and helical when the site of release precesses.

Urokinase-type plasminogen activator is effective in fibrin clearance in the absence of its receptor or tissue-type plasminogen activator.

The availability of gene-targeted mice deficient in the urokinase-type plasminogen activator (uPA), urokinase receptor (uPAR), tissue-type plasminogen activator (tPA), and plasminogen permits a critical, genetic-based analysis of the physiological and pathological roles of the two mammalian plasminogen activators. We report a comparative study of animals with individual and combined deficits in uPAR and tPA and show that these proteins are complementary fibrinolytic factors in mice. Sinusoidal fibrin deposits are found within the livers of nearly all adult mice examined with a dual deficiency in uPAR and tPA, whereas fibrin deposits are never found in livers collected from animals lacking uPAR and rarely detected in animals lacking tPA alone. This is the first demonstration that uPAR has a physiological role in fibrinolysis. However, uPAR-/-/tPA-/- mice do not develop the pervasive, multi-organ fibrin deposits, severe tissue damage, reduced fertility, and high morbidity and mortality observed in mice with a combined deficiency in tPA and the uPAR ligand, uPA. Furthermore, uPAR-/-/tPA-/- mice do not exhibit the profound impairment in wound repair seen in uPA-/-/tPA-/- mice when they are challenged with a full-thickness skin incision. These results indicate that plasminogen activation focused at the cell surface by uPAR is important in fibrin surveillance in the liver, but that uPA supplies sufficient fibrinolytic potential to clear fibrin deposits from most tissues and support wound healing without the benefit of either uPAR or tPA.

Therapeutic effects and possible mechanisms of a snake venom preparation in the fibrotic rat liver

The effects of a Chinese snake venom preparation from Agkistrodon halys pallas, used for treatment of hepatic fibrosis/cirrhosis in China, was investigated in an {in vivo} rat model and using in situ hepatic perfusion. Four groups were used in the experiments: (i) healthy, (ii) healthy/venom-treated, (iii) carbon tetrachloride (CCl4)-treated, and (iv) CCl4/venom-treated. Treatment effects were assessed by determining hepatic histopathology, biochemistry and fibrosis index parameters, bile production, biliary taurocholate recovery, hepatic mRNA expression of four bile salt transporters (Ntcp, Bsep, Oatp-1, and Oatp-3), comparison of hepatic microcirculation, fibrinolytic activity, and antithrombotic effects. Liver histopathology, biochemistry, and fibrosis index showed a dramatic improvement in venom-treated animals. There were significant differences in bile production between healthy/venom-treated and all other experimental groups and between CCl4/venom-treated and CCl4-treated animals, but no significant differences were found between CCl4/venom-treated and healthy animals. Biliary taurocholate recovery was significantly increased in healthy/venom-treated and CCl4/venom-treated animals. The expression of mRNA levels of the four bile salt transporters showed an increase after venom treatment. The hepatic microcirculation studies showed normalized sinusoidal beds in CCl4/venom-treated animals compared to healthy animals, whereas CCl4-treated animals showed abnormal profiles to the healthy and the CCl4/AHPV-treated animals. The fibrinogen and plasma thromboxane B-2 levels of healthy rats decreased with increasing dose after venom treatment. It was concluded that snake venom treatment may be therapeutic in treatment of hepatic fibrosis/cirrhosis by possibly a combination of increased bile flow and improved hepatic microcirculation, changes in bile salt transporter expression, and fibrinolytic and antithrombotic effects of the snake venom preparation.

Recombinant human activated protein C improves pulmonary function in ovine acute lung injury resulting from smoke inhalation and sepsis

Objective: To investigate the effects of recombinant human activated protein C (rhAPC) on pulmonary function in acute lung injury (ALI) resulting from smoke inhalation in association with a bacterial challenge. Design: Prospective, randomized, controlled, experimental animal study with repeated measurements. Setting: Investigational intensive care unit at a university hospital. Subjects: Eighteen sheep (37.2 +/- 1.0 kg) were operatively prepared and randomly allocated to either the sham, control, or rhAPC group (n = 6 each). After a tracheotomy had been performed, ALI was produced in the control and rhAPC group by insufflation of 4 sets of 12 breaths of cotton smoke. Then, a 30 mL suspension of live Pseudomonas aeruginosa bacteria (containing 2-5 x 10(11) colony forming units) was instilled into the lungs according to an established protocol. The sham group received only the vehicle, i.e., 4 sets of 12 breaths of room air and instillation of 30 mL normal saline. The sheep were studied in the awake state for 24 hrs and were ventilated with 100% oxygen. RhAPC (24 mu g/kg/hr) was intravenously administered. The infusion was initiated 1 hr post-injury and lasted until the end of the experiment. The animals were resuscitated with Ringer's lactate solution to maintain constant pulmonary artery occlusion pressure. Measurements and Main Results., In comparison with nontreatment in controls, the infusion of rhAPC significantly attenuated the fall in PaO2/FiO(2) ratio (control group values were 521 +/- 22 at baseline [BL], 72 +/- 5 at 12 hrs, and 74 +/- 7 at 24 hrs, vs. rhAPC group values of 541 +/- 12 at BL, 151 +/- 29 at 12 hours [p < .05 vs. control], and 118 +/- 20 at 24 hrs), and significantly reduced the increase in pulmonary microvascular shunt fraction (Qs/Qt; control group at BL, 0.14 +/- 0.02, and at 24 hrs, 0.65 +/- 0.08; rhAPC group at BL, 0.24 +/- 0.04, and at 24 hrs, 0.45 +/- 0.02 [p < .05 vs. control]) and the increase in peak airway pressure (mbar; control group at BL, 20 +/- 1, and at 24 hrs, 36 +/- 4; rhAPC group at BL, 21 +/- 1, and at 24 hrs, 28 +/- 2 [p < .05 vs. control]). In addition, rhAPC limited the increase in lung 3-nitrotyrosine (after 24 hrs [%]: sham, 7 +/- 2; control, 17 +/- 1; rhAPC, 12 +/- 1 [p < .05 vs. control]), a reliable indicator of tissue injury. However, rhAPC failed to prevent lung edema formation. RhAPC-treated sheep showed no difference in activated clotting time or platelet count but exhibited less fibrin degradation products (1/6 animals) than did controls (4/6 animals). Conclusions. Recombinant human activated protein C attenuated ALI after smoke inhalation and bacterial challenge in sheep, without bleeding complications.

Resistance of porcine blood clots to lysis relates to poor activation of porcine plasminogen by tissue plasminogen activator

In-vitro experimentation was performed on porcine and human blood to determine their comparative responsiveness to a novel fibrinolytic inhibitor and thereby assess whether the pig is a suitable animal model for subsequent in-vivo testing of this inhibitor. Thromboelastography showed the clots formed from porcine whole blood to be highly resistant to tissue plasminogen activator (t-PA)-catalyzed lysis, and this communication offers the resistance of porcine plasminogen to activation by t-PA as an explanation. Porcine blood containing 100 and 1500 IU/ml added t-PA lysed very slowly, having LY30 values of 1.9 +/- 1.4 and 2.9 +/- 1.9%, respectively. In contrast, the LY30 values for the human clots containing 100 and 1500 IU/ml t-PA were 77.1 +/- 6.3 and 93.3 +/- 1.3%, respectively. Moreover, purified porcine plasminogen was activated very slowly by added t-PA in the presence of both human and porcine fibrin. Activation of plasminogen by the endogenous activators, as measured by the euglobulin clot lysis time, was greatly prolonged for the pig (22 +/- 3 h) compared with the human (3.5 +/- 1.5 h). These results suggest caution in using the pig as an experimental model when studying the effects of various agents on fibrinolysis.

Comparison of active venom components between Eastern brown snakes collected from South Australia and Queensland

The abundance and activity of the prothrombin activator (pseutarin C) within the venom of the Eastern brown snake (Pseudonaja textilis textilis) is the primary determinant of its coagulation potency. Textilinin-1, also in this venom, is a plasmin inhibitor which is thought to exert its toxic effects through the slowing of fibrinolysis. The aim of this report is to determine if there are differences in the potency of the venom from Eastern brown snakes collected from South Australia (SA) compared to those from Queensland (QLD). A concentration of 0.4 mu g/ml venom protein from six QLD specimens clotted citrated plasma in an average time of 21.4 +/- 3.3 s compared to 68.7 +/- 2.4 s for the same amount of SA venom (averaged for six individuals). The more potent procoagulant activity of the QLD venom was measured between 0.4 and 94 mu g/ml venom protein in plasma. The anti-plasmin activity of textilinin was also greater in the venom of the snakes collected from QLD, causing full inhibition of plasmin at approximately 1.88 mu g/ml of venom protein compared to approximately 7.5 mu g/ml for the SA venoms. It is concluded that geographic differentiation of the Eastern brown snakes results in significant differences venom potency.

Diagnosis of canine bleeding disorders

Canine bleeding disorders arise due to a multitude of conditions and require detailed clinical and laboratory investigation. A productive diagnostic approach depends on a thorough patient history, physical examination, haemostatic screening tests and an array of specific diagnostic tests. Patient history is necessary to assist determination of the onset, severity and possible aetiologies of a bleeding disorder. Similarly, a complete physical examination should ideally allow differentiation between disorders of primary and secondary haemostasis. Following this distinction, a variety of laboratory tests are indicated to further define the nature of the bleeding episode. These tests may be broadly categorised as screening tests of primary haemostasis, secondary haemostasis and fibrinolysis, and specific tests directed at identifying particular disorders. Appropriate utilisation of these tests and interpretation of their results in conjunction with patient signalment, history and clinical signs affords the greatest chance of a successful diagnosis.

Polyphosphate as a haemostatic modulator

Funding This work was supported by the British Heart Foundation [grant number FS/11/2/28579]. © 2016 Authors; published by Portland Press Limited.

Novel aspects of platelet factor XIII function

Acknowledgments The study was supported by grants FS/11/2/28579 (N.J.M.) from the British Heart Foundation and the University of Aberdeen Development Trust.

Functional characterization of SjB10, an intracellular serpin from Schistosoma japonicum

Serine protease inhibitors (serpin) play essential roles in many organisms. Mammalian serpins regulate the blood coagulation, fibrinolysis, inflammation and complement activation pathways. In parasitic helminths, serpins are less well characterized, but may also be involved in evasion of the host immune response. In this study, a Schistosoma japonicum serpin (SjB10), containing a 1212 bp open reading frame (ORF), was cloned, expressed and functionally characterized. Sequence analysis, comparative modelling and structural-based alignment revealed that SjB10 contains the essential structural motifs and consensus secondary structures of inhibitory serpins. Transcriptional profiling demonstrated that SjB10 is expressed in adult males, schistosomula and eggs but particularly in the cercariae, suggesting a possible role in cercarial penetration of mammalian host skin. Recombinant SjB10 (rSjB10) inhibited pancreatic elastase (PE) in a dose-dependent manner. rSjB10 was recognized strongly by experimentally infected rat sera indicating that native SjB10 is released into host tissue and induces an immune response. By immunochemistry, SjB10 localized in the S. japonicum adult foregut and extra-embryonic layer of the egg. This study provides a comprehensive demonstration of sequence and structural-based analysis of a functional S. japonicum serpin. Furthermore, our findings suggest that SjB10 may be associated with important functional roles in S. japonicum particularly in host-parasite interactions.