scholarly journals Highly Procoagulant Extracellular Vesicles in Amniotic Fluid

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4961-4961
Author(s):  
Johannes Thaler ◽  
Lena Hell ◽  
Lukas Wisgrill ◽  
Andreas Spittler ◽  
Michael Schwameis ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Amniotic Fluid ◽  
Blood Coagulation ◽  
Extracellular Vesicles ◽  
Whole Blood ◽  
Blood Clotting ◽  
Factor Xa ◽  
Coagulation Cascade ◽  
Contact Activation ◽  
Plasma Factor

Abstract Background: The pathomechanisms underlying disseminated intravascular coagulation (DIC) following amniotic fluid (AF) embolism remain to be fully elucidated. Highly procoagulant phosphatidylserine (PS)- and tissue factor (TF) expressing extracellular vesicles (EVs) might play a central role. Objective: To perform extensive analyses of the procoagulant properties of AF with a panel of functional coagulation assays and flow cytometry to investigate the pathogenesis of AF induced DIC. Methods: A prothrombinase assay, an EV-TF dependent factor Xa (FXa) generation assay, a modified thrombin- and fibrin-generation assay, a whole blood clotting model and flow cytometry were applied in AF- and control plasma. Results: Phosphatidylserine expression was 21-fold increased in AF compared to plasma. Factor Xa generation was extremely high when TF-expressing EVs from AF were co-incubated with recombinant FVIIa. In the thrombin- and fibrin generation assay AF-derived EVs strongly activated the blood coagulation cascade via PS and TF. In a whole blood clotting model AF-derived TF-expressing EVs significantly shortened the clotting time from 734 ± 139 seconds in the presence- to 232 ± 139 seconds in the absence of an anti-TF antibody. The contact activation pathway via factor FXII was not affected. Applying flow cytometry, a sub-population of PS- and TF co-expressing EVs was clearly identified in AF. Conclusions: We thoroughly investigated the effect of AF on blood coagulation and found that PS+ and TF+ EVs determine its procoagulant potential. Taken together our data further delineate the pathomechanisms underlying AF-induced coagulopathy, which could improve diagnostic- and treatment modalities. Disclosures No relevant conflicts of interest to declare.

The Haemocompatibility of Biomaterials In Vitro: Investigations on the Mechanism of the Whole-blood Clot Formation Test

1992 ◽  
Vol 20 (3) ◽  
pp. 390-395 ◽  
Author(s):  
Thomas Groth ◽  
Katrin Derdau ◽  
Frank Strietzel ◽  
Frank Foerster ◽  
Hartmut Wolf
Keyword(s):  
Whole Blood ◽  
Blood Clotting ◽  
Blood Clot ◽  
Formation Rate ◽  
Clot Formation ◽  
Coagulation Cascade ◽  
Contact Activation ◽  
Human Fibrinogen ◽  
Static Conditions

Twenty years ago Imai & Nose introduced a whole-blood clotting test for the estimation of haemocompatibility of biomaterials in vitro In our paper a modification of this assay is described and the mechanism of clot formation further elucidated. It was found that neither the inhibition of platelet function nor the removal of platelets from blood significantly changed the clot formation rate on glass and polyvinyl chloride in comparison to the rate tor whole blood. Scanning electron microscopy demonstrated that platelets were not involved in clot formation near the blood/biomaterial interface. Thus, it was concluded that the system of contact activation of the coagulation cascade dominates during clot formation under static conditions. The latter conclusion was supported by the fact that preadsorption of human serum albumin or human fibrinogen onto the glass plates used, decreased the clot formation rate in the same manner.

Two parallel prothrombin activator systems in Australian rough-scaled snake, Tropidechis carinatus

2005 ◽  
Vol 93 (01) ◽  
pp. 40-47 ◽  
Author(s):  
Md. Abu Reza ◽  
Sanjay Swarup ◽  
Manjunatha Kini
Keyword(s):  
Blood Coagulation ◽  
Blood Clotting ◽  
Cdna Sequence ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Venom Gland ◽  
Factor X ◽  
Specific Expression ◽  
Life Saving ◽  
Sequence Encoding

SummaryIt is uncommon for similar pathways/systems to be involved in highly divergent functions within single organisms. Earlier, we have shown that trocarin D, a venom prothrombin activator, from the Australian rough-scaled snake Tropidechis carinatus, is structurally and functionally similar to the blood coagulation factor Xa (FXa). The presence of a haemostatic system in these snakes implies that they have two parallel prothrombin activating systems: one in the plasma, that participates in the life saving process of blood clotting and the other in their venom, where it acts as a toxin. Here, we report the complete cDNA sequence encoding the blood coagulation factor X (FX) from the liver of T. carinatus. Deduced T. carinatus FX sequence shows ~80% identity with trocarin D but ~50% identity with the mammalian FX. Our present study confirms the presence of two separate genes – one each for FX and trocarin D, that code for similar proteins in T. carinatus snake. These two genes have different expression sites and divergent uses suggesting that snake venom prothrombin activators have probably evolved by the duplication of the liver FX gene and subsequently marked for tissue-specific expression in the venom gland.

Effects of Tannins fromGeumjaponicumon the Catalytic Activity of Thrombin and Factor Xa of Blood Coagulation Cascade

1998 ◽  
Vol 61 (11) ◽  
pp. 1356-1360 ◽  
Author(s):  
Hui Dong ◽  
Shao-Xing Chen ◽  
R. Manjunatha Kini ◽  
Hong-Xi Xu
Keyword(s):  
Catalytic Activity ◽  
Blood Coagulation ◽  
Factor Xa ◽  
Coagulation Cascade

scholarly journals Role of the blood coagulation cascade in hepatic fibrosis

2018 ◽  
Vol 315 (2) ◽  
pp. G171-G176 ◽  
Author(s):  
Asmita Pant ◽  
Anna K. Kopec ◽  
James P. Luyendyk
Keyword(s):  
Blood Coagulation ◽  
Hepatic Fibrosis ◽  
Animal Studies ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Primary Source ◽  
Bleeding Risk ◽  
Normal Function ◽  
Coagulation Cascade ◽  
Rapid Progression

Liver is the primary source of numerous proteins that are critical for normal function of the blood coagulation cascade. Because of this, diseases of the liver, particularly when affiliated with severe complications like cirrhosis, are associated with abnormalities of blood clotting. Although conventional interpretation has inferred cirrhosis as a disorder of uniform bleeding risk, it is now increasingly appreciated as a disease wherein the coagulation cascade is precariously rebalanced. Moreover, prothrombotic risk factors are also associated with a more rapid progression of fibrosis in humans, suggesting that coagulation proteases participate in disease pathogenesis. Indeed, strong evidence drawn from experimental animal studies indicates that components of the coagulation cascade, particularly coagulation factor Xa and thrombin, drive profibrogenic events, leading to hepatic fibrosis. Here, we concisely review the evidence supporting a pathologic role for coagulation in the development of liver fibrosis and the potential mechanisms involved. Further, we highlight how studies in experimental animals may shed light on emerging clinical evidence, suggesting that beneficial effects of anticoagulation could extend beyond preventing thrombotic complications to include reducing pathologies like fibrosis.

Dual Inhibition of Blood Coagulation Factors Xa and Iia Synergize To Reduce Thrombus Weight and Thrombin Generation in a Rabbit Arteriovenous Shunt Model of Thrombosis.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1866-1866
Author(s):  
Thomas B. McClanahan ◽  
Sangita M. Baxi ◽  
Liguo Chi ◽  
Tawny Dahring ◽  
Weston R. Gould ◽  
...  
Keyword(s):  
Blood Coagulation ◽  
Single Molecule ◽  
Thrombin Generation ◽  
Ex Vivo ◽  
Factor Xa ◽  
Coagulation Cascade ◽  
Vehicle Group ◽  
Thrombin Inhibitor ◽  
Antithrombotic Effect ◽  
Dual Inhibitor

Abstract Several compounds currently in development for the treatment of thrombotic disorders demonstrate high levels of specificity for single targets of the blood coagulation cascade such as factor Xa and thrombin. However, development of a single molecule dual inhibitor against factor Xa and thrombin may expand the efficacy to safety ratio of treatment options for arterial and venous thrombosis. The objective of this study was to determine if simultaneous administration of PD 0313052, a selective Xa inhibitor and argatroban, a direct thrombin inhibitor, would lead to a synergistic antithrombotic effect in a rabbit AV shunt model of thrombosis. Intravenous administration of PD 0313052 alone at doses of 0.1, 0.3, and 1.0 mg/kg/min resulted in thrombus weight (TW) reductions of 11±3, 25±10 and 67±7 % compared to the vehicle group. Argatroban at 1, 3 and 10 mg/kg/min reduced TW 16±13, 47±10 and 75±6 %. When PD 0313052 was administered at 0.1 mg/kg/min in combination with argatroban at 1, 3 or 10 mg/kg/min TW was reduced 50±7, 60±7 and 82±9 %. Likewise, argatroban at 1 mg/kg/min combined with 0.1, 0.3 or 1mg/kg/min of PD 0313052 resulted in TW reductions of 56±9, 60±9 and 84±5 %, respectively. At the lowest combined doses of PD 0313052 and argatroban there was no change in bleeding time relative to the additive fold-increases from each drug alone. The EC50 of intravenously administered PD 0313052 and argatroban was 67±23 and 178±58 ng/ml, respectively. When the drugs were combined the EC50 was reduced to 12±6 ng/ml with the PD 0313052/argatroban combination and to 83±29 ng/ml with the argatroban/PD 0313052 combination. A synergistic effect was also observed in an ex vivo assay of thrombin generation (TG). Predicted additive inhibition of TG based on the individual effects of each compound was −9±7, 9±2 and 29±7 % compared to 10±5, 32±5 and 55±3 % with the 313052/argatroban combination. The predicted effects of the argatroban/PD 0313052 combination was −9±7, 1±7 and 16±9 % compared to the actual inhibition of 5±3, 14±5 and 31±7 %. These results demonstrate a significant synergistic antithrombotic effect by combining low doses of a factor Xa and a thrombin inhibitor and support the hypothesis that development of a single molecule inhibitor against different hemostatic targets may offer greater efficacy in the prevention and treatment of venous and arterial thrombosis.

Influence of Nanopolymers with Different End-Functionalities on Platelet Function and the Coagulation Cascade - An Ex-Vivo Study.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4038-4038
Author(s):  
Meera Chitlur ◽  
Erin Ware ◽  
Sujata Kannan ◽  
Wendy Hollon ◽  
Steve Buck ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Platelet Aggregation ◽  
Surface Charge ◽  
Platelet Function ◽  
Whole Blood ◽  
Clot Formation ◽  
Coagulation Cascade ◽  
Cancer Drug ◽  
Dendritic Polymers ◽  
Drug Concentrations

Abstract Dendritic polymers are branched nanopolymers with a central core and multiple peripheral functional groups that offer great potential as high payload delivery vehicles carrying multiple copies of drug molecules, targeting ligands and imaging agents to their site of action. Their nanoscopic dimensions offer exciting possibilities for achieving high intracellular drug concentrations in many therapeutic areas including anti-cancer drug delivery. Biocompatibility and biodistribution of dendritic polymers may be influenced by surface charge and concentration. One of the major challenges in their use is the effect on coagulation. The objective of this study was to determine the effect of change in surface charge and concentration of dendritic polymer on cellular and enzymatic components of coagulation. Materials and Methods: The effect of increasing concentrations (1, 10, 100, and 1000mcg/ml) of polyamidoamine (PAMAM) dendrimers with -COOH (anionic), -OH (neutral), and -NH2 (cationic) end functionalities, on platelet function and coagulation was evaluated using thromboelastography, whole blood aggregation, and flow cytometry. The thromboelastographic profile and platelet aggregation studies were obtained on samples of whole blood incubated for thirty minutes with dendrimer. Platelets were incubated with FITC labelled dendrimer for 30,60 and 120 mins, to determine uptake and platelet activation using flow cytometry. All tests were performed in triplicate. RESULTS: Thromboelastography: No significant effect on clot formation (time to clot formation and size) was seen with PAMAM-COOH (COOH) or PAMAM-OH (OH). Prolonged time to initiation of clot and decreased size were noted with 100 and 1000mcg/ml of PAMAM-NH2(NH2) as shown in figure1, indicating impairment of both the enzymatic and cellular components of the coagulation system. Whole Blood Aggregation: Neither platelet aggregation nor secretion were significantly affected by COOH or OH. Platelet aggregation was significantly decreased with NH2 at 100 and 1000mcg/ml. Flow Cytometry: Spontaneous CD62 activation was seen in platelets incubated with NH2. No spontaneous CD62 activation was noted with COOH or OH even at 1000mcg/ml. Platelet uptake of FITC labeled dendrimer was assessed at 30, 60 and 120mins of incubation. Increased uptake of FITC labeled dendrimer was noted at 2 hours with NH2. TEG clotting Profiles with PAMAM-NH2. TEG clotting Profiles with PAMAM-NH2. CONCLUSIONS: Surface charge of the dendritic nanopolymers plays a significant role on its effect on coagulation and platelet function. The anionic -COOH terminated and neutral -OH terminated dendrimers had no effect on hemostasis even at the highest concentrations while the cationic-NH2 was associated with inhibition of platelet aggregation and delayed clot initiation at higher concentrations. This would indicate that the anionic and neutral dendrimers would serve as better vehicles than cationic dendrimers for targeted delivery of therapeutic agents.

A Zymogen-like Factor Xa Improves Hemostasis in a Murine Bleeding Model

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1476-1476 ◽  
Author(s):  
Jasuja Reema ◽  
Sunita Patel-Hett ◽  
Rodney M. Camire ◽  
Joachim Fruebis ◽  
Debra Pittman
Keyword(s):  
Blood Loss ◽  
Whole Blood ◽  
Thrombin Generation ◽  
Ex Vivo ◽  
Factor Xa ◽  
Coagulation Cascade ◽  
Effective Control ◽  
Normal Male ◽  
Severe Bleeding ◽  
K Value

Abstract In many clinical indications, effective control of bleeding is needed. Factor Xa (FXa) is a vitamin K-dependent trypsin-like serine protease that interacts with non-enzymatic coagulation factor Va (FVa) on negatively charged membrane surfaces to generate thrombin during hemostasis. Based on its central role in the coagulation cascade at the intersection of both intrinsic and extrinsic pathways, direct administration of FXa is an attractive approach to restoring hemostasis in bleeding disorders by leading to direct thrombin generation and fibrin formation. However, the short plasma half-life of the activated FXa protease renders it inadequate as a therapeutic for acute bleeding. Here, we investigate FXaI16L,a recently described variant of coagulation FXa engineered to overcome these limitations. The FXaI16L variant has an isoleucine (I) to leucine (L) substitution at amino acid 16 (based on chymotrypsin numbering). FXaI16L exhibits zymogen-like properties with both reduced activity and sensitivity toward plasma inhibitors. In the presence of its cofactor, FVa, FXaI16L activity is restored. We assessed the hemostatic activity of FXaI16L in an acute tail bleeding model that results in severe bleeding in normal mice. Ex vivo pharmacodynamic parameters in plasma and whole blood were also measured. FXaI16L was administrated intravenously to normal male C57BL/6J mice at doses of 1, 10, 25, 50, 100, or 200 μg/kg. Control mice received vehicle only. Two minutes post administration, a 3 mm tail transection was made. Tails were immediately immersed in tubes containing pre-warmed phosphate buffered saline for blood collection over a ten minute period. Bleeding times were recorded and volume of blood loss was determined by measurement of the hemoglobin content in the collected blood. Following administration of FXaI16L, a dose dependent reduction in bleeding was observed. Mice dosed with FXaI16Lshowed a decrease in blood loss of 12% (1 μg/kg), 16.6% (10 μg/kg), 26.7% (25 μg/kg), 45.3% (50 μg/kg), 62.9% (100 μg/kg), and 69.6% (200 μg/kg) compared to vehicle-dosed mice. The estimated ED50 was 46 μg/kg. Following infusion of FXaI16L (25 μg/kg) or vehicle into normal male CD-1 mice, we measured the ex vivo activity in plasma using an activated partial thromboplastin time (aPTT) clotting assay and a thrombin generation assay (TGA). Plasma collected from FXaI16L-dosed animals at 2 minutes post-injection displayed a 67% reduction in aPTT compared to vehicle-dosed mice. Dosing of FXaI16L at 25 μg/kg also enhanced thrombin generation, as reflected by a shortened lag phase, increased peak thrombin, increased endogenous thrombin potential and higher velocity index compared to vehicle treated mice. We also measured thromboelastography (TEG) parameters of whole blood collected from mice infused with FXaI16L. At a 10 μg/kg intravenous dose of FXaI16L, the TEG R-value and K-value measures of clotting time decreased, while TEG alpha angle and maximum amplitude increased compared to vehicle treated mice. We conclude that administration of FXaI16L in normal mice enhances hemostasis, decreasing bleeding in an injury model. Together, these studies suggest that FXaI16L may provide a new and unique way to achieve hemostasis in clinical situations of uncontrolled bleeding. Disclosures Reema: Pfizer: Employment. Patel-Hett:Pfizer: Employment. Camire:Pfizer: Consultancy, Patents & Royalties, Research Funding. Fruebis:Pfizer: Employment. Pittman:Pfizer: Employment.

scholarly journals Effect of Lidocaine and Epinephrine on Human Erythrocyte Shape and Vesiculability of Blood Cells

2015 ◽  
Vol 2015 ◽  
pp. 1-10 ◽  
Author(s):  
Tanja Slokar ◽  
Carlos Lopez-Mariscal ◽  
Judita Lea Krek ◽  
Roman Štukelj ◽  
Oskar Zupanc ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Extracellular Vesicles ◽  
Whole Blood ◽  
Blood Cells ◽  
Statistical Power ◽  
Erythrocyte Shape ◽  
Plasma Lidocaine ◽  
Average Factor ◽  
Shape Changes

The effect of local anesthetic composed of lidocaine and epinephrine on vesiculability of blood cells and erythrocyte shape was studied. Whole blood and plasma were incubated with lidocaine/epinephrine. Extracellular vesicles were isolated by centrifugation and washing and counted by flow cytometry. Lidocaine/epinephrine and each component alone were added to diluted blood. Shape changes were recorded by micrographs. An ensemble of captured frames was analyzed for populations of discocytes, echinocytes, and stomatocytes by using statistical methods. Incubation of whole blood and blood plasma with lidocaine/epinephrine considerably increased concentration of extracellular vesicles in isolates (for an average factor 3.4 in blood and 2.8 in plasma). Lidocaine/epinephrine caused change of erythrocyte shape from mainly discocytic to mainly stomatocytic (higher than 50%). Lidocaine alone had even stronger stomatocytic effect (the percent of stomatocytes was higher than 95%) while epinephrine had echinocytic effect (the percent of echinocytes was higher than 80%). The differences were highly statistically significantp<10-8with statistical powerP=1. Lidocaine/epinephrine induced regions of highly anisotropically curved regions indicating that lidocaine and epinephrine interact with erythrocyte membrane. It was concluded that lidocaine/epinephrine interacts with cell membranes and increases vesiculability of blood cellsin vitro.

scholarly journals CYTOTOXICITY AND HEMOCOMPATIBILITY OF DOXORUBICIN-LOADED PLGA NANOPARTICLES

2020 ◽  
Vol 19 (1) ◽  
pp. 71-80
Author(s):  
Yu. A. Malinovskaya ◽  
E. I. Kovalenko ◽  
T. S. Kovshova ◽  
N. S. Osipova ◽  
O. O. Maksimenko ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Blood Coagulation ◽  
Hemoglobin Concentration ◽  
Coagulation Cascade ◽  
Coagulation System ◽  
Intracranial Tumour ◽  
Glioblastoma Cells ◽  
Human Glioblastoma ◽  
Human Glioblastoma Cells

Introduction. The use of polymeric biodegradable nanoparticles (NP) as drug delivery systems is a promising approach to overcome histohematomatic barriers. Thus, poloxamer 188-coated poly (lactide-co-glycolide) (PLGA) NP are able to overcome blood-brain barrier and to deliver therapeutic agents, in particular doxorubicin, into intracranial tumour upon intravenous administration. It is important to evaluate NP interaction with blood components in preclinical studies.The objective of the study was to investigate cytotoxicity and hemocompatibility of doxorubicin-loaded PLGA NP (Dox-PLGA NP), to essess NP uptake by glioblastoma cells.Materials and methods. The influence of NP on coagulation cascade was evaluated by prothrombin time measuring before and after plasma incubation with NP. To assess NP thrombogenicity the platelet activation level was determined by flow cytometry. The NP hemolytic activity (released hemoglobin concentration) was measured spectrophotometrically. NP cytotoxicity was determined by MTS assay. NP uptake by human glioblastoma cells was evaluated by flow cytometry.Results. Dox-PLGA NP did not influence blood coagulation time and thrombocyte activity at concentrations up to 100 mcg/mL: PT values were 12–15 s for all tested samples, and P-selectin expression level did not exceed 15 %. All samples were not hemolytic after 3 h of incubation. Cytotoxicity of doxorubicin released from PLGA NP on glioma U87MG cells was comparable to that of free doxorubicin. As shown by flow cytometry Dox-PLGA NP were efficiently internalized into the cells.Conclusion. The study of hemocompatibility confirmed the safety of Dox-PLGA NP: NP did not influence blood coagulation system and did not induce hemolysis. NP were efficiently internalized into the human glioblastoma cells and produced considerable antitumor effect in vitro.

THE CHANGES OF FACTOR VIII ANTIGEN DURING THE COAGULATION PROCESS

1987 ◽  
Author(s):  
I Tanaka ◽  
A Yoshioka ◽  
T Fujiwara ◽  
H Nakai ◽  
H Fukui
Keyword(s):  
Monoclonal Antibodies ◽  
Serum Level ◽  
Factor Viii ◽  
Blood Coagulation ◽  
Whole Blood ◽  
Initial Phase ◽  
Blood Clotting ◽  
Coagulation Process ◽  
The Mean

The changes of factor VIII (F. VIII) during blood coagulation process is still controversial. We analyzed the F. VIII antigen (F. VIII:Ag) at various intervals of in vitro blood clotting by immunoassays using polyclonal and different kinds of monoclonal antibodies to F. VIII.We used two immunoassays, an immunoradiometric assay (IRMA) and an enzyme-linked immunosorbent assay (ELISA). The IRMA was performed by the method of Peake et al. using high-titer allo-antibodies to F. VIII. The ELISA was performed by two-site solid phase system consisting of alloantibodies as the first and one of three kinds of monoclonal antibodies (NMC-VIII/1, -VIII/2 or C5*) as the second antibody. Using the immunoblotting technique, it had been shown that NMC-VIII/1 recognized the 80/79 kDa derived from C-terminus and both NMC-VIII/2 and C5 recognized 54 kDa derived from N-terminus.The mean levels of F. VIII:Ag in 20 normal plasmas and sera were 0.97±0.23 U/ml and 0.68±0.21 U/ml, respectively using the polyclonal IRMA. The mean levels of F. VIII:Ag in normal sera were 0.14±0.05 U/ml (NMC-VIII/1), 0.71±0.21 U/ml (NMC-VIII/2), and 0.012±0.02 U/ml (C5) using the monoclonal ELISAs. In the initial phase of whole blood coagulation in vitro, the increase of F. VIII: Ag was observed by the polyclonal IRMA as F. VIII:C assayed by a one-stage clotting method increased. On the other hand, the F. VIII:Ag assayed by NMC-VIII/1 or C5 monoclonal ELISA progressively decreased to the serum level within 30 min. The F. VIII:Ag by NMC-VIII/2 declined to the serum level at a slow rate. In order to study the influence of thrombin on F. VIIIrAg during blood clotting, a synthesized selective thrombin inhibitor (MD-805, Mitsubishi Chemical Ind.) was previously added to the whole blood tested. The changes of F. VIII:Ag with MD-805 by the monoclonal ELISAs were almost the same as those without MD-805.It is suggested that in the whole blood coagulation process the antigenicity of F. VIII molecule changes in the initial phase (within 30 min.), but that thrombin does not play the main role of the phenomenon in physiological concentration.*C5 was kindly supplied from Dr. C. Fulcher (Scripps Clinic and Research Foundation, USA)

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