Improved AP-3 production through combined ARTP mutagenesis, fermentation optimization, and subsequent genome shuffling.

Ansamitocin (AP-3) is an ansamycins antibiotic isolated from Actinosynnema pretiosum and demonstrating high anti-tumor activity. To improve AP-3 production, the A. pretiosum ATCC 31565 strain was treated with atmospheric and room temperature plasma (ARTP). Four stable mutants were obtained by ARTP, of which the A. pretiosum L-40 mutant produced 242.9 mg/L AP-3, representing a 22.5% increase compared to the original wild type strain. With seed medium optimization, AP-3 production of mutant L-40 reached 307.8 mg/L; qRT-PCR analysis revealed that AP-3 biosynthesis-related gene expression was significantly up-regulated under optimized conditions. To further improve the AP-3 production, genome shuffling (GS) technology was used on the four A. pretiosum mutants by ARTP. After three rounds of GS combined with high-throughput screening, the genetically stable recombinant strain G3-96 was obtained. The production of AP-3 in the G3-96 strain was 410.1 mg/L in shake flask cultures, which was 44.5% higher than the L-40 production from the parental strain, and AP-3 was increased by 93.8% compared to the wild-type A. pretiosum. These results suggest that the combination of mutagenesis, seed medium optimization, and GS technology can effectively improve the AP-3 production capacity of A. pretiosum and provide an enabling methodology for AP-3 industrial production.

Ellipsoidal mesoscale mineralization pattern in human cortical bone revealed in 3D by plasma focused ion beam serial sectioning.

Visualizing bone mineralization and collagen fibril organization at intermediate scales between the nanometer and the hundreds of microns range, is still an important challenge. Similarly, visualizing cellular components which locally affect the tissue structure requires a precision of a few tens of nanometers at maximum while spanning several tens of micrometers. In the last decade, gallium focused ion beam (FIB) equipped with a scanning electron microscope (SEM) proved to be an extremely valuable structural tool to meet those ends. In this study, we assess the capability of a recent plasma FIB-SEM technology which provides a potential increase in measurement speed over gallium FIB-SEM, thus paving the way to larger volume analysis. Nanometer-scale layers of demineralized and mineralized unstained human femoral lamellar bone were sequentially sectioned over volumes of 6-16,000 µm3. Analysis of mineralized tissue revealed prolate ellipsoidal mineral clusters measuring approximately 1.1 µm in length by 700 nm at their maximum diameter. Those features, suggested by others in high resolution studies, appear here as a ubiquitous motif in mineralized lamellar bone over thousands of microns cubed, suggesting a heterogeneous and yet regular pattern of mineral deposition past the single collagen fibril level. This large scale view retained sufficient resolution to visualize the collagen fibrils while also partly visualizing the lacuno-canalicular network in three-dimensions. These findings are strong evidence for suitability of PFIB as a bone analysis tool and the need to revisit bone mineralization over multi-length scales with mineralized tissue.

Group O plasma as a media supplement for CAR-T cells and other adoptive T-cell therapies.

BACKGROUND: Most chimeric antigen receptor T (CAR-T) cells and other adoptive T-cell therapies (ACTs) are currently manufactured by ex vivo expansion of patient lymphocytes in culture media supplemented with human plasma from group AB donors. As lymphocytes do not express A or B antigens, the isoagglutinins of non-AB plasmas are unlikely to cause deleterious effects on lymphocytes in culture. STUDY DESIGN AND METHODS: Seeding cultures with peripheral blood mononuclear cell (PBMNC) concentrates from group A1 donors and using a CAR-T culture protocol, parallel cultures were performed, each with unique donor plasmas as media supplements (including group O plasmas with high-titer anti-A and group AB plasmas as control). An additional variable, a 3% group A1 red blood cell (RBC) spike, was added to simulate a RBC-contaminated PBMNC collection. Cultures were monitored by cell count, viability, flow cytometric phenotype, gene expression analysis, and supernatant chemokine analysis. RESULTS: There was no difference in lymphocyte expansion or phenotype when cultured with AB plasma or O plasma with high-titer anti-A. Compared to controls, the presence of contaminating RBCs in lymphocyte culture led to poor lymphocyte expansion and a less desirable phenotype-irrespective of the isoagglutinin titer of the plasma supplement used. CONCLUSIONS: This study suggests that ABO incompatible plasma may be used as a media supplement when culturing cell types that do not express ABO antigens-such as lymphocytes for CAR-T or other ACT. The presence of contaminating RBCs in culture was disadvantageous independent of isoagglutinin titer.

Thrombin Generation in Fresh and Frozen-Thawed Platelet Poor Plasma - Is there a Difference?

BACKGROUND: Applicability of thrombin generation tests to clinical routine has been sought for many years. The aim of this study was to compare thrombin generation measured in fresh platelet poor plasma (f-PPP) and frozen-thawed platelet poor plasma (ft-PPP) to prove the consistency of results. METHODS: In this prospective study, thrombin generation was measured in twenty-fold repetitions in 3.2% citrate PPP obtained from male healthy blood donors aged 19 - 39 years (n = 54 donations). The tests were performed with fresh PPP and repeated after storing the PPP at -60°C. In two subgroup analyses, the effect of higher and lower normal baseline platelet counts on the Calibrated Automated Thrombogram (CAT) assay and the influence of ABO blood groups on thrombin generation were analyzed. RESULTS: Referring to the parameters of thrombin generation most frequently used in studies, peak thrombin of f-PPP and ft-PPP agreed in about 50% of the samples. Endogenous thrombin potential (ETP) of f-PPP and ft-PPP agreed in nearly two-thirds of the samples. A slightly but significantly slower kinetic was found in the thrombin generation of ft-PPP compared with f-PPP. At least in f-PPP, ETP correlates with baseline platelet counts of the whole blood sample. Peak thrombin was significantly higher in non-O blood groups compared to O blood group. CONCLUSIONS: A low level of agreement between the results of f-PPP and ft-PPP is shown. In terms of practicability of sample collection using semi-automated thrombin-generation assays ft-PPP should be preferred over f-PPP. We therefore recommend using ft-PPP in clinical studies.

Venom-Induced Consumption Coagulopathy Following Hump-Nosed Pit Viper (Genus: Hypnale) Envenoming in Sri Lanka: Uncertain Efficacy of Fresh Frozen Plasma.

INTRODUCTION: Hump-nosed pit vipers (Hypnale spp) cause the highest number of venomous snakebites in Sri Lanka. Bites commonly cause local envenoming leading to local pain, swelling, and necrosis of the site of the bite. Acute kidney injury is the most common systemic manifestation, and some patients develop venom-induced consumption coagulopathy (VICC). Genus Hypnale comprises 3 species. Of them, H hypnale is found in Sri Lanka and the Western Ghats region of India. The other 2 (H nepa and H zara) are endemic species in Sri Lanka. METHODS: This study included 500 patients with hump-nosed viper bites studied prospectively over 4.5 y starting June 2014. All patients were assessed and the data were collected by the principal investigator (primary data). A subgroup of patients who developed VICC is described. There were 2 groups, including proven (patients with the specimen of the snake) and probable (specimen of snake not available) bites. RESULTS: Thirty (n=500; 6%) patients developed VICC; of them, 17 (3%) were proven cases, and 13 (2%) were probable cases. In both groups, 24 (80%) recovered, 2 (7%) progressed to chronic kidney disease, 1 (3%) died of severe hemostatic dysfunction, and 3 (10%) were lost to follow-up. Systemic bleeding was observed in 16 patients (53%), including hematuria (microscopic and gross) in 8 (27%) and venipuncture bleeding in 5 (17%). Eleven (37%) developed local bleeding at the site of the bite. Fresh frozen plasma was administered to 20 patients (67%), among whom only 11 (55%) experienced early correction of VICC. In both groups, 15 (50%) developed acute kidney injury, and 2 (7%) progressed to chronic kidney disease. Microangiopathic hemolysis was observed in 18 patients (60%) and thrombocytopenia in 16 (53%). Thrombotic microangiopathy was detected in 13 patients (43%), of whom 10 (33%) developed hemolytic uremic syndrome and 2 (7%) had thrombotic thrombocytopenic purpura. Of patients with VICC in the proven group, 94% (n=16) was caused by H hypnale and 1 (6%) was caused by H zara. In the proven group, median international normalized ratio was 3.7 (interquartile range 1.6-5.0); in the probable group, it was 5.0 (interquartile range 2.1-5.4). CONCLUSIONS: We found that 6% of patients develop hemostatic dysfunction after hump-nosed viper bites. However, which patients will develop coagulopathy or die of envenoming is unpredictable. Reliable and accessible treatments are unmet essential needs because antivenoms for these bites are currently not available in the country. Therapy with fresh frozen plasma has doubtful efficacy in early correction of VICC and needs further evaluation.

Allogeneic platelet-rich plasma (PRP) is superior to platelets or plasma alone in stimulating fibroblast proliferation and migration, angiogenesis, and chemotaxis as relevant processes for wound healing.

BACKGROUND: Platelet-rich plasma (PRP) is frequently used in the treatment of acute and chronic wounds. One of the major problems concerning the use of PRP is the absence of a well-characterized and standardized product, which leads to a high variety in study outcomes. Therefore, more studies on the composition and standardization of PRP in wound healing are needed. STUDY DESIGN AND METHODS: Platelet concentrates derived from healthy blood donors were made in plasma (PC-plasma) or platelet additive solution (PC-PAS). The effects of PC-plasma, PC-PAS, and plasma were then tested on proliferation, differentiation, and migration of fibroblasts, as well as sprouting of endothelial cells in fibrin gels and chemotaxis of white blood cells (WBCs). RESULTS: PC-plasma stimulates the migration and proliferation of human dermal fibroblasts more than plasma or platelets alone. Furthermore, platelet factors decrease the expression of α-smooth muscle actin in dermal fibroblast cultures. PC-plasma also stimulates sprouting of endothelial cells. Finally, PC-plasma also acts as a strong chemoattractant for WBCs. CONCLUSIONS: Allogeneic PC-plasma has beneficial effects on various aspects of wound healing in vitro and is superior to plasma or platelets alone. PC-plasma is an attractive candidate for further in vivo evaluation.

Disinfection and Sterilization Using Plasma Technology: Fundamentals and Future Perspectives for Biological Applications.

Recent studies have shown that plasma can efficiently inactivate microbial pathogens such as bacteria, fungi, and viruses in addition to degrading toxins. Moreover, this technology is effective at inactivating pathogens on the surface of medical and dental devices, as well as agricultural products. The current practical applications of plasma technology range from sterilizing therapeutic medical devices to improving crop yields, as well as the area of food preservation. This review introduces recent advances and future perspectives in plasma technology, especially in applications related to disinfection and sterilization. We also introduce the latest studies, mainly focusing on the potential applications of plasma technology for the inactivation of microorganisms and the degradation of toxins.

Factor XIII: a coagulation factor with multiple plasmatic and cellular functions.

Factor XIII (FXIII) is unique among clotting factors for a number of reasons: 1) it is a protransglutaminase, which becomes activated in the last stage of coagulation; 2) it works on an insoluble substrate; 3) its potentially active subunit is also present in the cytoplasm of platelets, monocytes, monocyte-derived macrophages, dendritic cells, chondrocytes, osteoblasts, and osteocytes; and 4) in addition to its contribution to hemostasis, it has multiple extra- and intracellular functions. This review gives a general overview on the structure and activation of FXIII as well as on the biochemical function and downregulation of activated FXIII with emphasis on new developments in the last decade. New aspects of the traditional functions of FXIII, stabilization of fibrin clot, and protection of fibrin against fibrinolysis are summarized. The role of FXIII in maintaining pregnancy, its contribution to the wound healing process, and its proangiogenic function are reviewed in details. Special attention is given to new, less explored, but promising fields of FXIII research that include inhibition of vascular permeability, cardioprotection, and its role in cartilage and bone development. FXIII is also considered as an intracellular enzyme; a separate section is devoted to its intracellular activation, intracellular action, and involvement in platelet, monocyte/macrophage, and dendritic cell functions.

Flow-Induced Transitions of Red Blood Cell Shapes under Shear.

A recent study of red blood cells (RBCs) in shear flow [Lanotte et al., Proc. Natl. Acad. Sci. U.S.A. 113, 13289 (2016)PNASA60027-842410.1073/pnas.1608074113] has demonstrated that RBCs first tumble, then roll, transit to a rolling and tumbling stomatocyte, and finally attain polylobed shapes with increasing shear rate, when the viscosity contrast between cytosol and blood plasma is large enough. Using two different simulation techniques, we construct a state diagram of RBC shapes and dynamics in shear flow as a function of shear rate and viscosity contrast, which is also supported by microfluidic experiments. Furthermore, we illustrate the importance of RBC shear elasticity for its dynamics in flow and show that two different kinds of membrane buckling trigger the transition between subsequent RBC states.

How viscoelastic is human blood plasma?

Blood plasma has been considered a Newtonian fluid for decades. Recent experiments (Brust et al., Phys. Rev. Lett., 2013, 110) revealed that blood plasma has a pronounced viscoelastic behavior. This claim was based on purely elastic effects observed in the collapse of a thin plasma filament and the fast flow of plasma inside a contraction-expansion microchannel. However, due to the fact that plasma is a solution with very low viscosity, conventional rotational rheometers are not able to stretch the proteins effectively and thus, provide information about the viscoelastic properties of plasma. Using computational rheology and a molecular-based constitutive model, we predict accurately the rheological response of human blood plasma in strong extensional and constriction complex flows. The complete rheological characterization of plasma yields the first quantitative estimation of its viscoelastic properties in shear and extensional flows. We find that although plasma is characterized by a spectrum of ultra-short relaxation times (on the order of 10-3-10-5 s), its elastic nature dominates in flows that feature high shear and extensional rates, such as blood flow in microvessels. We show that plasma exhibits intense strain hardening when exposed to extensional deformations due to the stretch of the proteins in its bulk. In addition, using simple theoretical considerations we propose fibrinogen as the main candidate that attributes elasticity to plasma. These findings confirm that human blood plasma features bulk viscoelasticity and indicate that this non-Newtonian response should be seriously taken into consideration when examining whole blood flow.

Tear osmolarity is sensitive to exercise-induced fluid loss but is not associated with common hydration measures in a field setting.

This investigation (i) examined changes in tear osmolarity in response to fluid loss that occurs with exercise in a field setting, and (ii) compared tear osmolarity with common field and laboratory hydration measures.  Sixty-three participants [age 27.8 ± 8.4 years, body mass 72.15 ± 10.61 kg] completed a self-paced 10 km run outside on a predetermined course. Body mass, tear fluid, venous blood and urine samples were collected immediately before and after exercise.  Significant (p < 0.001) reductions in body mass (1.71 ± 0.44%) and increases in tear osmolarity (8 ± 15 mOsm.L-1), plasma osmolality (7 ± 8 mOsm.kg-1), and urine specific gravity (0.0014 ± 0.0042 g.mL-1; p = 0.008) were observed following exercise. Pre- to post-exercise change in tear osmolarity was not significantly correlated (all p > 0.05) with plasma osmolality (rs = 0.24), urine osmolality (rs = 0.14), urine specific gravity (rs = 0.13) or relative body mass loss (r = 0.20).  Tear osmolarity is responsive to exercise-induced fluid loss but does not correlate with the changes observed using other common measures of hydration status in the field setting. Practitioners shouldn't directly compare or replace other common hydration measures with tear osmolarity in the field. ABBREVIATIONS: BML: Body Mass Loss; CV: Coefficient of Variation; Posm: Plasma osmolality; SD: Standard Deviation; Tosm: Tear Osmolarity; Uosm: Urine Osmolality; USG: Urine Specific Gravity; WBGT: Wet bulb globe thermometer.

The prognostic value of dynamic contrast-enhanced MRI contrast agent transfer constant Ktrans in cervical cancer is explained by plasma flow rather than vessel permeability.

BACKGROUND: The microvascular contrast agent transfer constant Ktrans has shown prognostic value in cervical cancer patients treated with chemoradiotherapy. This study aims to determine whether this is explained by the contribution to Ktrans of plasma flow (Fp), vessel permeability surface-area product (PS), or a combination of both. METHODS: Pre-treatment dynamic contrast-enhanced MRI (DCE-MRI) data from 36 patients were analysed using the two-compartment exchange model. Estimates of Fp, PS, Ktrans, and fractional plasma and interstitial volumes (vp and ve) were made and used in univariate and multivariate survival analyses adjusting for clinicopathologic variables tumour stage, nodal status, histological subtype, patient age, tumour volume, and treatment type (chemoradiotherapy vs radiotherapy alone). RESULTS: In univariate analyses, Fp (HR=0.25, P=0.0095) and Ktrans (HR=0.20, P=0.032) were significantly associated with disease-free survival while PS, vp and ve were not. In multivariate analyses adjusting for clinicopathologic variables, Fp and Ktrans significantly increased the accuracy of survival predictions (P=0.0089). CONCLUSIONS: The prognostic value of Ktrans in cervical cancer patients treated with chemoradiotherapy is explained by microvascular plasma flow (Fp) rather than vessel permeability surface-area product (PS).

Ultrastructural, Confocal and Viscoelastic Characteristics of Whole Blood and Plasma After Exposure to Cadmium and Chromium Alone and in Combination: An Ex Vivo Study.

BACKGROUND/AIMS: Heavy metal pollution is increasing in the environment, contaminating water, food and air supplies. This can be linked to many anthropogenic activities. Heavy metals are absorbed through the skin, inhalation and/or orally. Irrespective of the manner of heavy metal entry in the body, the blood circulatory system is potentially the first to be affected following exposure and adverse effects on blood coagulation can lead to associated thrombotic disease. Although the plasma levels and the effects of cadmium (Cd) and chromium (Cr) on erythrocytes and lymphocytes have been described, the environmental exposure to heavy metals are not limited to a single metal and often involves metal mixtures, with each metal having different rates of absorption, different cellular, tissue, and organ targets. Therefore the aim of this study is to investigate the effects of the heavy metals Cd and Cr alone and whether Cr synergistically increases the effect of Cd on physiological important processes such as blood coagulation. METHODS: Human blood was exposed to the heavy metals ex vivo, and thereafter morphological analysis was performed with scanning electron- and confocal laser scanning microscopy (CLSM) in conjunction with thromboelastography®. RESULTS: The erythrocytes, platelets and fibrin networks presented with ultrastructural changes, including varied erythrocytes morphologies, activated platelets and significantly thicker fibrin fibres in the metal-exposed groups. CLSM analysis revealed the presence of phosphatidylserine on the outer surface of the membranes of the spherocytic erythrocytes exposed to Cd and Cr alone and in combination. The viscoelastic analysis revealed only a trend that indicates that clots that will form after heavy metal exposure, will likely be fragile and unstable especially for Cd and Cr in combination. CONCLUSION: This study identified the blood as an important target system of Cd and Cr toxicity.

14-Day thawed plasma retains clot enhancing properties and inhibits tPA-induced fibrinolysis.

BACKGROUND: Plasma-first resuscitation attenuates trauma-induced coagulopathy (TIC); however, the logistics of plasma-first resuscitation require thawed plasma (TP) be readily available due to the obligatory thawing time of fresh frozen plasma (FFP). The current standard is storage of TP for up to 5 days at 4°C, based on factor levels at outdate, for use in patients at risk for TIC, but there remains a 2.2% outdated wastage rate. However, the multitude of plasma proteins in attenuating TIC remains unknown. We hypothesize that TP retains the ability to enhance clotting and reduce tPA-induced fibrinolysis at 14-day storage. METHODS: FFP was thawed and stored at 4°C at the following intervals: 14, 10, 7, 5, 3, and 1-day prior to the experiment. Healthy volunteers underwent blood draws followed by 50% dilution with TP stored at previously mentioned intervals as well as FFP, normal saline (NS), albumin, and whole blood (WB) control. Samples underwent tPA-modified (75 ng/mL) thrombelastography (TEG) with analysis of R-time, angle, maximum amplitude (MA), and LY30. RESULTS: TEG properties did not change significantly over the thawed storage. 14-day TP retained the ability to inhibit tPA-induced hyperfibrinolysis (median LY30% 9.6%) similar to FFP (5.6%), WB (14.6%), and superior to albumin (59.3%) and NS (58.1%). 14-day TP also retained faster clot formation (median angle, 66.2°) and superior clot strength (MA, 61.5 mm) to albumin (34.8°, 21.6 mm) and NS (41.6°, 32.2 mm). CONCLUSIONS: TP plasma stored for 14 days retains clot-enhancing ability and resistance to clot degradation similar to FFP. A clinical trial is needed to validate these in vitro results.

Red blood cell transfusion is associated with further bleeding and fresh-frozen plasma with mortality in nonvariceal upper gastrointestinal bleeding.

BACKGROUND: Blood products are commonly transfused for patients with nonvariceal upper gastrointestinal bleeding (NVUGIB). While concerns exist about further bleeding and mortality in subsets of patients receiving red blood cell (RBC) transfusion, the impact of non-RBC blood products has not previously been systematically investigated. The aim of the study was to investigate the associations between blood products transfusion, further bleeding, and mortality after acute NVUGIB. STUDY DESIGN AND METHODS: A retrospective cohort study examined further bleeding and 30-day and 1-year mortality in adult patients who underwent gastroscopy for suspected acute NVUGIB between 2008 and 2010 in three tertiary hospitals in Western Australia. Survival analysis was performed. RESULTS: A total of 2228 adults (63% male) with 2360 hospital admissions for NVUGIB met the inclusion criteria. Median age at presentation was 70 years (range, 19-99 years). Thirty-day mortality was 4.9% and 1-year mortality was 13.9%. Transfusion of 4 or more units of RBCs was associated with greater than 10 times the odds of further bleeding in patients with a hemoglobin level of more than 90 g/L (odds ratio, 11.9; 95% confidence interval [CI], 3.1-45.7; p ≤ 0.001), but was not associated with mortality. Administration of 5 or more units of fresh-frozen plasma (FFP) was associated with increased 30-day (hazard ratio, 2.8; 95% CI, 1.3-5.9; p = 0.008) and 1-year (hazard ratio, 2.6; 95% CI, 1.3-5.0; p = 0.005) mortality after adjusting for coagulopathy, comorbidity, Rockall score, and other covariates. CONCLUSION: In this large, multicenter study of NVUGIB, RBC transfusion was associated with further bleeding but not mortality, while FFP transfusion was associated with increased mortality in a subset of patients.

Effects of riboflavin and amotosalen photoactivation systems for pathogen inactivation of fresh-frozen plasma on fibrin clot structure.

BACKGROUND: Fresh-frozen plasma (FFP) transfusion carries a risk of viral transmission from donor to recipient. Riboflavin (Mirasol) and amotosalen (Intercept) are two pathogen inactivation (PI) methods that may enhance the safety of FFP for transfusion. Our study investigated the effects of Mirasol and Intercept treatment on fibrin formation and clot structure. STUDY DESIGN AND METHODS: FFP underwent either Mirasol or Intercept treatment, and aliquots were taken before addition of the compound, before illumination (after addition of compound only), and after treatment (addition of compound plus illumination). All samples underwent turbidimetric analysis, lysis analysis, assessment of clot permeation, and analysis by laser scanning confocal microscopy. RESULTS: After treatment, there was a decrease in optical density of the fibrin network for Mirasol and Intercept, lag time to fibrin formation was prolonged for Mirasol and lysis time for Intercept, clot permeability was significantly decreased, and clot density was increased for both. CONCLUSIONS: Our study shows that plasma treated with Mirasol and Intercept produces denser clots consisting of thinner fibers and warrants further studies to evaluate the clinical significance of these structural changes in fibrin clot formation.

Study of plasma coagulation induced by contact with calcium chloride solution.

Blood coagulation capability is one of the most important factors for the diagnosis of patients with thrombosis. Regarding the blood coagulation as an example of gelation of soft matter, we can apply an analytical method to this phenomenon and pick up some relevant parameters. In various systems, gelation dynamics induced by contact between a polymer solution and a crosslinker solution are well explained by the "moving boundary picture" (Yamamoto et al., J. Phys. Chem. B, 2010, 114, 10002-10009). The aim of this paper is to clarify whether this picture can be applied to a clinically important biological system used for blood coagulation tests. We have measured the time course of the thickness of a plasma gel layer formed when plasma comes in contact with calcium chloride solution in a rectangular cell and analyzed theoretically on the basis of the moving boundary picture. The entire process was well expressed using a scaled equation involving three parameters characterizing the plasma, k, Kin, and ß, where k is the time required to reach the incipient stage of three-dimensional network formation, the parameter Kin is proportional to calcium chloride concentration and ß is a constant. These results indicate the direct applicability of the general theory of gelation dynamics induced by contact between two solutions to the in vitro coagulation (gelation) of plasma, and the fitting parameters may be used for diagnosis.

Southern copperhead venom enhances tissue-type plasminogen activator induced fibrinolysis but does not directly lyse human plasma thrombi.

In addition to degrading fibrinogen as a source of consumptive coagulopathy, purified fractions of southern copperhead (Agkistrodon contortrix contortrix; A. c. contortrix) venom has been demonstrated to enhance fibrinolysis. The goal of this investigation was to characterize the kinetic fibrinolytic profile of A. c. contortrix venom in the absence and presence of tissue-type plasminogen activator (tPA) to determine if intact venom had tPA independent fibrinolytic properties. Utilizing thrombelastographic methods, the coagulation and fibrinolytic kinetic profiles of human plasma exposed to A. c. contortrix venom (0-6 µg/ml) were determined in the absence or presence of tPA (0-100 IU/ml). Then, plasma was exposed to 0-6 µg/ml of venom without tPA added and coagulation observed for 3 h. Venom significantly prolonged the onset of coagulation, decreased the velocity of thrombus growth but did not significantly decrease clot strength. In the presence of tPA, venom significantly decreased clot strength, shortened the time of onset of fibrinolysis, decreased clot lysis time but did not significantly affect the maximum rate of lysis. Lastly, while venom exposure in the absence of tPA significantly prolonged the onset of coagulation and decreased the velocity of clot growth, venom exposure did not result in detectable fibrinolysis over the 3 h observation period. A. c. contortrix venom enhances tPA mediated fibrinolysis by degrading plasma coagulation kinetics. Intact A. c. contortrix venom does not possess sufficient fibrinolytic activity to cause fibrinolysis in human plasma at the concentration tested.