Human Prothrombin Research Articles

Sequence Determinants of Mouse Protein C Interaction with Mouse Endothelial Protein C Receptor

The Endothelial Protein C Receptor (EPCR) is an important component of the Protein C anticoagulant pathway. The interaction of Protein C (PC) through its Gla domain with EPCR enhances PC activation, thus down-regulating thrombin production. EPCR can also bind human activated Factor VII (FVIIa) and modulate its activity and localization. The residues involved in receptor recognition in both PC and FVIIa are Phe4 and Leu8, located in the first portion of the Gla domain. The importance of Phe8 is indicated by the lack of EPCR binding of a PC variant that contains a Val8 from human prothrombin. Remarkably, the similarity between PC and FVIIa for EPCR binding is lacking in the mouse. Others and we have shown that mouse FVIIa (mFVIIa, which contains a Leu4 and a Leu8) interacts with mouse EPCR (mEPCR) poorly, thus failing to model the spectrum of known human FVIIa properties. In previous work, we generated mFVIIa chimeras that contain parts of the mouse PC (mPC) Gla domain and determined that three residues in the mPC Gla domain can confer mEPCR binding to mFVIIa. Specifically, molecule mFVIIa-FMR that contained the Leu4->Phe, Leu8->Met and Trp9->Arg from mPC was functionally similar to mFVIIa and could bind mEPCR as a true gain-of-function. However, little is known on the contribution of any/all of these positions in mPC binding to mEPCR. Here, we wanted to understand the sequence determinants that dictate this interaction. For this, we generated single amino acid mutants of mPC at position 4, 8 or 9 from the corresponding residues of mFVIIa. Using conditioned medium from transiently transfected cells, we tested the ability of each mPC mutant to bind to mEPCR expressed on the surface of CHO-K1 cells. A single substitution of Phe4 with Leu abolished mEPCR binding of mPC, in contrast to modifications at position 8 (Met to Leu) or 9 (Arg to Trp). The importance of Phe4 for the mPC-mEPCR interaction was confirmed in a reverse experiment modifying mFVIIa (that has poor mEPCR affinity) individually at position 4 (Leu to Phe), 8 (Leu to Met) or 9 (Trp to Arg) according to the mPC sequence. We found that Leu4->Phe was the sole modification that could confer mEPCR binding to mFVIIa. We have previously shown that the interaction of mFVIIa-FMR with mEPCR enhances its hemostatic function (vs. mFVIIa) after administration in hemophilic mice that have undergone injury (Pavani G et al, Blood 2014). To further explore the contribution of position 4 (Leu->Phe [L4F]) in these effects, recombinant mFVIIa-L4F was purified. Titration of mFVIIa-L4F on CHO-K1 cells expressing mEPCR showed a specific and dose-dependent receptor binding, in contrast to mFVIIa, confirming our previous data (see above). Moreover, mFVIIa-L4F showed no difference in clotting activity compared to mFVIIa in a prothrombin time-based assay. In order to compare mFVIIa-L4F to mFVIIa in its ability to generate mouse thrombin, we used a thrombin generation assay using hemophilia B plasma spiked with either procoagulant. We found that addition of either mFVIIa or mFVIIa-L4F generated similar amounts of thrombin at all concentrations tested (3.1 - 25 nM). Therefore, mFVIIa-L4F exhibited similar coagulant activity to mFVIIa but gained mEPCR binding capacity, a feature shared with mFVIIa-FMR. Further experiments are underway to determine whether the single substitution in mFVIIa-L4F is sufficient to recapitulate the enhanced hemostatic properties observed in vivo with mFVIIa-FMR. In conclusion, our findings identify a single amino acid residue (Phe4) in the Gla domain of mouse PC that plays a critical role in the binding to its natural receptor. This property can also be transplanted into mFVIIa, without affecting its coagulant activity. These observations reveal another difference between human and mouse systems and may have implications for EPCR-dependent functions or properties of other vitamin K-dependent proteins. DisclosuresNo relevant conflicts of interest to declare.

Use of Four-Factor Prothrombin Complex Concentrate in the Emergency Department: A Review

Warfarin, a vitamin K antagonist (VKA), is the mainstay for the prevention and treatment of arterial and venous thrombosis. The complication associated with VKA therapy that causes the most concern is the risk of major bleeding events, particularly anticoagulant-associated intracranial hemorrhage. 1 Hanger HC Geddes AA Wilkinson TJ Lee M Baker AE Warfarin related intracerebral heamorrhage: better outcomes when reversal includes prothrombin complex concentrates. Intern Med J. 2012; 43: 308-316 Crossref Scopus (42) Google Scholar A variety of approaches may be used to assist with reversal of the anticoagulation effects of VKAs; these approaches often combine vitamin K with prothrombin complex concentrate (PCC), fresh frozen plasma (FFP), or recombinant factor VIIa. Human 4-factor PCC (4 F-PCC [Kcentra], CSL Behring, King of Prussia, PA) was the first treatment approved by the Food and Drug Administration for the rapid reversal of VKA therapy. 2 Kcentra [package insert]. CSL Behring LLC, Kankakee, IL2013 Google Scholar 4 F-PCC restores clotting factors II, VII, IX, and X, protein C, and protein S, which are needed for blood hemostasis and are depleted with VKA therapy. Further studies will likely evaluate the role of 4 F-PCC in the management of trauma-related massive bleeding, as well as for reversal of the new oral factor Xa and direct thrombin inhibitors. This review provides an updated assessment of the use of 4 F-PCC in the management of VKA reversal in the emergency department. Courtney Willis is 2015 PharmD Candidate, University of Florida College of Pharmacy, Gainesville, FL. Brad Hall is Clinical Pharmacy Specialist–Emergency Medicine and PGY2 Emergency Medicine Pharmacy Director, Lakeland Regional Medical Center, Lakeland, FL. Use of Four-Factor Prothrombin Complex Concentrate in the Emergency DepartmentJournal of Emergency NursingVol. 41Issue 3PreviewAs a doctoral candidate, I have conducted extensive research on vitamin K antagonist (VKA) reversal using human 4-factor prothrombin complex concentrate (4-FPCC), compared with traditional reversal using fresh frozen plasma (FFP). I found the article “Use of Four-Factor Prothrombin Complex Concentrate in the Emergency Department: A Review” by Willis and Hall1 to be comprehensive, thorough, and an overall excellent review. Although existing research has yet to demonstrate improved clinical outcomes with 4-FPCC, the current body of evidence regarding hemostatic efficacy, international normalized ratio (INR) correction time profiles, and thromboembolitic complications was well reported. Full-Text PDF

CP-072 Use of human prothrombin complex concentrate in patients at high risk of severe bleeding in a trauma hospital

BackgroundSevere deficiency in blood clotting factors is a cause of massive haemorrhage whose management in emergency situations is the subject of debate.PurposeA prospective, observational study, to ascertain how prothrombin complex...

Meizothrombin Preparation and Its Role in Fibrin Formation and Platelet Aggregation

Meizothrombin (MT) is one of prothrombin derivatives which appears in haemostasis activation area. However, its role in haemostasis regulation isn’t clear. We studied the role of MT in fibrin formation, platelet activation and aggregation. A new effective method of obtaining MT from native human prothrombin was developed using immobilised prothrombin activator from Echis multisquamatis venom. The protein was stable and electrophoretically pure. Platelet-rich plasma for aggregation study and gel-sieved platelets for flow-cytometry were separated from blood of healthy donors. It was shown that MT transformed fibrinogen to fibrin and activated clotting factor XIII. MT didn’t activate gel-sieved intact platelets, but in platelet-rich plasma, increased platelet aggregation induced by ADP, collagen and adrenalin.

Process-scale purification and analytical characterization of highly gamma-carboxylated recombinant human prothrombin

Prothrombin (coagulation Factor II) is a complex multidomain glycoprotein that plays a central role in blood coagulation. It is the zymogen precursor to the protease thrombin that catalyzes the formation of the fibrin clot and regulates a multitude of other cellular responses related to coagulation and hemostasis. For the biological activity of prothrombin, the vitamin K dependent posttranslational modification of glutamic acid residues to gamma-carboxylglutamic acid is of crucial importance. Prothrombin can be recombinantly expressed using mammalian cell culture. However, the product is a heterogeneous mixture of variants with different degrees of carboxylation, requiring separation of closely related charge isoforms. A second challenge for purification is the need to remove traces of the product-related impurity thrombin, a protease, to extremely low levels. In this work, we describe a purification strategy that provides solutions to both challenges and results in an efficient and robust process for active recombinant prothrombin. We also describe the analytical characterization of recombinant prothrombin by HPLC, LC–MS/MS, and complementary biochemical assays.

Validation of a commercially available kit to detect anti-phosphatidylserine/prothrombin antibodies in a cohort of systemic lupus erythematosus patients

Antiprothrombin antibodies detection comprises two different ELISAs: prothrombin coated on irradiated plates (aPT) or phosphatidylserine/prothrombin (aPS/PT) as the antigen. While several commercial kits are available for the detection of aPT, aPS/PT are usually detected by in-house assays. Recently, a new commercially available kit was launched and, therefore, we decided to test its efficiency by comparing it to our in-house assay. aPS/PT were tested by our in-house assay (aPS/PT(ih)) in 75 SLE patients, using Immulon 1 plates coated with phosphatidylserine, purified human prothrombin and 1%BSA-TBS-CaCl as blocking and diluents. Data from this assay were compared to those obtained by the QUANTA Lite aPS/PT screen, IgG and IgM Elisa (INOVA Diagnostics, Inc, San Diego, USA) commercial kits (aPS/PT(c)). aPS/PT were found in 41.3% and 46.7% of SLE patients by the aPS/PT(ih) and the aPS/PT(c), respectively. There was a positive correlation between IgG aPS/PT(ih) and aPS/PT(c) assays (R(2)=0. 861 by Spearman test, p=0.0027). Sensitivity and specificity for APS were 62.2% and 97.4% (AUC 0.780) for the aPS/PT(ih) assay and 70.3% and 84.2% (AUC 0.858) for the aPS/PT(C). Shorter running times were also seen when comparing the aPS/PT(ih) vs. aPS/PT(c) (7hours vs. 3hours, respectively). The aPS/PT(C) is a reproducible and accurate test for the detection of aPS/PT, bringing also the advantage of shorter running times.

Amino Acid Region 1000–1008 of Factor V Is a Dynamic Regulator for the Emergence of Procoagulant Activity

Single chain factor V (fV) circulates as an Mr 330,000 quiescent pro-cofactor. Removal of the B domain and generation of factor Va (fVa) are vital for procoagulant activity. We investigated the role of the basic amino acid region 1000-1008 within the B domain of fV by constructing a recombinant mutant fV molecule with all activation cleavage sites (Arg(709)/Arg(1018)/Arg(1545)) mutated to glutamine (fV(Q3)), a mutant fV molecule with region 1000-1008 deleted (fV(ΔB9)), and a mutant fV molecule containing the same deletion with activation cleavage sites changed to glutamine (fV(ΔB9/Q3)). The recombinant molecules along with wild type fV (fV(WT)) were transiently expressed in COS-7L cells, purified, and assessed for their ability to bind factor Xa (fXa) prior to and following incubation with thrombin. The data showed that fV(Q3) was severely impaired in its interaction with fXa before and after incubation with thrombin. In contrast, KD(app) values for fV(ΔB9) (0.9 nM), fVa(ΔB9) (0.4 nM), and fV(ΔB9/Q3) (0.7 nM) were similar to the affinity of fVa(WT) for fXa (0.3 nM). Two-stage clotting assays revealed that although fV(Q3) was deficient in its clotting activity, fV(ΔB9/Q3) had clotting activity comparable with fVa(WT). The kcat value of prothrombinase assembled with fV(ΔB9/Q3) was minimally affected, whereas the Km value of the reaction was increased 57-fold compared with the Km value obtained with prothrombinase assembled with fVa(WT). These findings strongly suggest that amino acid region 1000-1008 of fV is a regulatory sequence protecting the organisms from spontaneous binding to fXa and unnecessary prothrombinase complex formation, which in turn results in catastrophic physiological consequences.

Effects of the dilution rate on cell cycle distribution and PEI-mediated transient gene expression by CHO cells in continuous culture

In this study, a continuous culture system was applied to mammalian cells on large scale, and polyethyleneimine (PEI) mediated transient gene expression (TGE). PEI MAX 40,000 was chosen as a superior reagent from three types of PEI. The cell cycle distribution of cells in batch and continuous cultures was determined, in which the effects of cell cycle distribution on transfection efficiency, post-transfection proliferation and recombinant prothrombin expression were evaluated. Compared with cells from end-log and plateau phase in batch culture, cells from mid-log phase possessed a larger fraction of S and G2/M phase cells and a smaller fraction of G1 phase cells. In the continuous culture, the fraction of cells in the S and G2/M phases increased and the fraction of cells in the G1/G0 phase decreased with increasing dilution rates. Cells from the continuous culture run at highest dilution rate had excellent proliferation, transfection efficiency and protein expression. These results were confirmed by transfecting cells synchronized to different phases. The G2/M arrested cells exhibited a nearly 10-fold increase in recombinant human prothrombin production relative to that of non-dividing cells. The use of continuous culture for large scale transfection demonstrated a better cell physiological state for TGE process.

Antimicrobial, Antioxidant and Anticoagulation Activities of Korean Radish (Raphanus sativus L.) Leaves

Radish (Raphanus sativus) is a common cruciferous vegetable, and its aerial parts, called Mu-chung in Korean, have plentiful nutritional components such as vitamins, minerals and dietary fibers. Mu-chung has been used as a kimchi, a traditional Korean fermented dish, and dried Mu-chung is an important component of soups commonly consumed during winter in Korea. Since the advent of the mass production of radish in Korea, with the segregation of farm areas and towns and changing diets, Mu-chung has mostly been discarded instead of utilized. In addition, studies concerning the efficient utilization and useful bioactivities of Mu-chung are still lacking worldwide. In this study, we prepared the ethanol extract of Mu-chung and its subsequent solvent fractions. Antimicrobial, antioxidation, and anticoagulation activities were then evaluated in the hopes of developing a functional biomaterial from Korean radishes' aerial parts. The ethanol extraction yield for hot-air dried Mu-chung was 5.6%, and the fraction yields of n-hexane (H), ethylacetate (EA), butanol (B) and water residue were 25.3, 3.6, 19.4, and 51.7%, respectively. Analysis of total polyphenol and total flavonoid contents showed that the EA fraction had the highest content (97.57 and 152.91 mg/g) amongst the fractions. In antimicrobial activity assays, the H and EA fractions were effective against gram positive bacteria (Staphylococcus aureus, Listeria monocytogenes, and Bacillus subtilis), but not effective against gram negative bacteria (Escherichia coli and Pseudomonas aeruginosa). The B fraction also exhibited moderate antibacterial activity, suggesting that the extract of Mu-chung has various antibacterial components. In antioxidation activity assays, the EA fraction showed strong DPPH, ABTS and nitrite scavenging activities ( of ), including reducing power. In anticoagulation activity assays, the EA fraction demonstrated strong inhibition activity against human thrombin and prothrombin. Prominent anticoagulation activity was found in aPTT assays; the aPTT of the EA fraction was extended 15-fold compared than that of the solvent control. Our results suggest that Mu-chung is an attractive nutritional food material possessing useful bioactivities, and the EA fraction of Mu-chung could be developed as a functional food ingredient.

Zastosowanie koncentratu czynników zespołu protrombiny w leczeniu zaburzeń hemostazy osoczowej po operacji transplantacji serca

Hemostasis involves complex processes meaning blood not to extravasate. It's temporary cessation is necessary to perform cardiac surgery procedure with cardiopulmonary bypass. In heart transplantation, standard procedures of heparine neutralization are insufficient. We present results obtained from the group of 10 patients (8 men and 2 women) in mean age of 41±15 years, who underwent heart transplantation procedure with Lower-Shumway technique in moderate hypothermia (28°C). Mean cardiopulmonary bypass time was 218±20min, cross clampling aortic time was 114±18min and organ cold ischemia time was 221±16min. Postoperative human prothrombin complex was given after INR result (2.2±0.3) was obtained followed by normalized activated clotting time (ACT) 124±17seconds. In high risk patients undergoing cardiac surgery procedure, standard international normalized ration (INR) measurements following ACT results are required to obtain more detailed information concerning plasma hemostasis disturbances. Prothromplex Total NF (Baxter International Inc, USA) is a safe option to correct the problem without risk of overloading the patients circulatory system.

Molecular Basis of Phospholipid Synergy in Promoting Blood Coagulation Reactions

Abstract 1109Most steps in the blood coagulation cascade obligatorily take place on membrane surfaces and are dependent on the exposure of phosphatidylserine (PS). Many coagulation proteins bind to PS-containing membrane bilayers in a calcium-dependent manner via gamma-carboxyglutamate-rich (GLA) domains. Furthermore, certain phospholipids, most notably phosphatidylethanolamine (PE), strongly synergize with PS to promote clotting reactions. We have recently been investigating the molecular basis of lipid-Ca2+ and lipid-GLA interactions in blood clotting using solid-state NMR (SSNMR) studies that employ isotopically labeled PS. Our studies have revealed that Ca2+ induces two predominant conformations of the PS headgroup (Boettcher et al., Biochem. 50:2264–73, 2011). We now report the quantification of the intermolecular and intramolecular atomic distances within Ca2+-induced PS nanoclusters using SSNMR. We have also expanded SSNMR studies to phospholipid bilayers composed of trinary mixtures of PS, PC, and PE as a means to further investigate the impact of PE on PS headgroup conformations in an intact lipid bilayer (PE-PS synergy). Initial results suggest that incorporation of PE into the bilayer alters the Ca2+: PS ratio required to fully saturate the L-serine headgroups of PS. Additionally, PE appears to affect the predominant PS headgroup conformations in the presence of Ca2+, and may induce a third L-serine conformation. SSNMR studies utilizing isotopically labeled PE headgroups are currently underway which should provide novel information concerning PE's interactions with PS, Ca2+, and/or GLA domains.Previous SSNMR studies from our lab have also indicated that a fraction of L-serine headgroups in PS adopt a new conformation when bovine prothrombin fragment 1 is bound to PS/PC bilayers (Tavoosi et al., J. Biol. Chem. 286:23247–53, 2011). These data support the idea that GLA domains contain an L-serine-specific binding site. We have now bound human prothrombin fragment 1 to membranes containing labeled PS headgroups for use in SSNMR studies. In addition, we have also employed surface plasmon resonance (SPR) experiments to investigate the interactions of both human and bovine prothrombin with bilayers containing PS and PE. These data demonstrate that binding affinities and binding footprints are similar for both prothrombin proteins, and that PE enhances binding affinities and increases the number of GLA binding sites for both proteins. Together, results from SSNMR and SPR studies suggest that different GLA domains use similar molecular mechanisms to interact with the membrane surface. Disclosures:No relevant conflicts of interest to declare.

Increased Activity of Prothrombinase Fgl-2 in Peripheral Blood Mononuclear Cells of Patients with B-Cell Lymphoma.

AbstractAbstract 2665 Background:Hematologic and solid tumors are associated with hypercoagulability the reason for which has not been delineated. Prothrombinase named fibrinogen-like protein 2 (FGL-2) is a 70 kD transmembrane protein that was found to have a quality of a serine protease capable of directly cleaving prothrombin to thrombin. FGL-2 is synthesized by monocytes, T-lymphocytes and endothelial cells. FGL-2 protein and its mRNA have been previously found within different tumor cells. Aim:To study the role of FGL-2 in patients with lymphoproliferative disorders. Our hypothesis is that upregulation of FGL-2 activity in patients with B-cell malignancies may contribute to tumorigenesis via generation of thrombin leading to increased angiogenesis and spread/metastasis of malignant cells. Methods:Thrombin generation reflecting FGL-2 activity was measured in homogenized peripheral blood mononuclear cells (PBMC) from 29 patients with active lymphoproliferative disorders and 107 normal controls. Informed consent was obtained from every participant. PBMC extracts were incubated with an equal volume of human prothrombin (final concentration10 μM) for 30 min at 37 °C. Thrombin generation was measured at 405 nm using an automated plate reader after addition of chromogen S-2238. The thrombin activity of each sample was calculated by comparison with absorbance curve generated by known concentrations of human thrombin. FGL-2 was immunoprecipitated (IP) from PBMC with an anti-FGL-2 antibody and EZview Red Protein A Affinity Gel (sigma # P6486). The activity of IP FGL-2 was measured by thrombin generation assay. The expression of FGL-2 was analyzed in HUVEC and PBMC in the presence or absence of IF-γ at 20 ng/ml. Total RNA was isolated using RNAqueous™ (Ambion #AM1912) and RT-PCR, analysis was performed using Rotor-gene RG-3000 (Corbett). The difference in cycle time (ΔCT) was measured by comparing FGL-2 gene with ABL-1 gene (house keeping gene). The relative quantification was calculated by the formula RQ= 2−ΔCT. HUVEC were transfected with 0.5 μM SiRNA synthesized complementary to FGL-2 (Target SiRNA) using Dharmacon ON-TARGET plus SMART pool reagent (Thermo Fisher Scientific) according to manufacturer instructions. FGL-2 expression following addition of target SiRNA was compared to that obtained following addition of non-specific (non-target) SiRNA. Student’s t-test was used for all comparisons. Results:As shown in the table, almost 3-fold increase in FGL-2 activity in PBMC was observed among patients with active B-cell lymphoma, either aggressive or indolent, as compared to that of healthy controls (p<0.0001 for all comparisons). No difference in FGL-2 activity in PBMC between patients with aggressive and indolent lymphoma was observed (p=0.68).Three-fold increase in thrombin generation was obtained in PBMC from the patients following IP, similarly to that observed in non-IP PBMC. A significant increase in mRNA of FGL-2 was found in either PBMC of lymphoma patient or HUVEC treated with IF-γ. In HUVEC the increase in FGL-2 mRNA was inhibited by 80% following treatment with specific SiRNA. Conclusions:1.There is increased activity of prothrombinase FGL-2 in PBMC of patients with B-cell lymphoma. Possible correlation between increased mRNA and protein activity of FGL-2 and B-cell lymphoma burden will be evaluated.2.The mechanistic effect of FGL-2 on tumorigenesisangiogenesis will be studied.Control subjects (N=107)Indolent B-cell lymphoma: FL, MCL, MZL, SLL (n=16)Aggressive B-cell lymphoma: DLBC, BL (n=13)All B-cell lymphoma (n=29)Mean ± SD (ng/ml)142.2 ± 118.4427.1 ± 219.2389.3 ± 267.5410.2 ± 238.3Range (ng/ml)0–439180–948153–1011153–1011P valueP<0.0001*P<0.0001*P<0.0001*FL – follicular lymphoma; MCL – mantle cell lymphoma; MZL – marginal cell lymphoma; SLL – small lymphocytic lymphoma; DLBCL – diffuse large B-cell lymphoma; BL – Burkitt’s lymphoma*compared to the control group. Disclosures:No relevant conflicts of interest to declare.

Association between Human Prothrombin Variant (T165M) and Kidney Stone Disease

We previously reported the association between prothrombin (F2), encoding a stone inhibitor protein - urinary prothrombin fragment 1 (UPTF1), and the risk of kidney stone disease in Northeastern Thai patients. To identify specific F2 variation responsible for the kidney stone risk, we conducted sequencing analysis of this gene in a group of the patients with kidney stone disease. Five intronic SNPs (rs2070850, rs2070852, rs1799867, rs2282687, and rs3136516) and one exonic non-synonymous single nucleotide polymorphism (nsSNP; rs5896) were found. The five intronic SNPs have no functional change as predicted by computer programs while the nsSNP rs5896 (c.494 C>T) located in exon 6 results in a substitution of threonine (T) by methionine (M) at the position 165 (T165M). The nsSNP rs5896 was subsequently genotyped in 209 patients and 216 control subjects. Genotypic and allelic frequencies of this nsSNP were analyzed for their association with kidney stone disease. The frequency of CC genotype of rs5896 was significantly lower in the patient group (13.4%) than that in the control group (22.2%) (P = 0.017, OR 0.54, 95% CI 0.32–0.90), and the frequency of C allele was significantly lower in the patient group (36.1%) than that in the control group (45.6%) (P = 0.005, OR 0.68, 95% CI 0.51–0.89). The significant differences of genotype and allele frequencies were maintained only in the female group (P = 0.033 and 0.003, respectively). The effect of amino-acid change on UPTF1 structure was also examined by homologous modeling and in silico mutagenesis. T165 is conserved and T165M substitution will affect hydrogen bond formation with E180. In conclusion, our results indicate that prothrombin variant (T165M) is associated with kidney stone risk in the Northeastern Thai female patients.

Fibrin formation by staphylothrombin facilitates Staphylococcus aureus-induced platelet aggregation

Interactions of Staphylococcus aureus (S. aureus) and platelets play an important role in the pathogenesis of intravascular infections such as infective endocarditis (IE). A typical feature of S. aureus is the ability to generate thrombin activity through the secretion of two prothrombin activating molecules, staphylocoagulase and von Willebrand factor-binding protein (vWbp), which bind to human prothrombin to form the enzymatically active staphylothrombin complex. The role of staphylothrombin in the interaction between S. aureus and platelets has not yet been studied. We found that in contrast with thrombin, staphylothrombin did not directly activate human platelets. However, the staphylothrombin-mediated conversion of fibrinogen to fibrin initiated platelet aggregation and secondary activation and facilitated S. aureus-platelet interactions. Both the genetic absence of staphylocoagulase and vWbp and pharmacological inhibition of staphylothrombin increased the lag time to aggregation, and reduced platelet trapping by S. aureus in high shear stress conditions. The combined inhibition of staphylothrombin and immunoglobulin binding to platelets completely abolished the ability of S. aureus to aggregate platelets in vitro. In conclusion, although staphylothrombin did not directly activate platelets, the formation of a fibrin scaffold facilitated bacteria-platelet interaction, and the inhibition of staphylothrombin resulted in a reduced activation of platelets by S. aureus.

High Resolution X-Ray Structure of Snake Venom Factor V: Evolution of a Hemostatic Cofactor to a Toxin Poised to Inflict Maximal Damage to Mammalian Blood Coagulation

Abstract 375Poisonous snakes frequently harbor activators of mammalian coagulation as part of the toxin repertoire in their venom. The venom of Pseudonaja textilis (Ptex, common brown snake) contains an efficient activator of human prothrombin comprised of a Xa-like protein tightly bound to a Va-like protein. The constituents of this complex exhibit high sequence homology to the corresponding activated coagulation factors in mammalian blood. Factors Xa and Va are produced in blood upon proteolytic activation of their precursors, complex with each other in membrane-dependent reactions to form prothrombinase and catalyze thrombin formation at the site of vascular damage. In contrast, the venom proteins are constitutively active, form a complex in solution and can efficiently catalyze prothrombin activation in the absence of membranes. These properties likely drive the disseminated and consumptive coagulopathy associated with evenomation by P. textilis. The Va-like component (Ptex-Va) of P. textilis venom is a single chain glycoprotein of 1430 residues with 53% identity to human factor V (hV) and a common A1-A2-B-A3-C1-C2 domain organization. The B-domain of Ptex-Va is significantly shorter than its counterpart in hV (46 vs. 836 residues). We now report a high resolution x-ray structure of recombinant Ptex-Va collected at 1.9 Å resolution and solved by molecular replacement. The resulting structure closely mimics those seen at lower resolution for inactivated bovine factor Va lacking the A2 domain and full length B-domainless human factor VIII (hVIII). Each A domain is formed by two cupredoxin-like β barrels with the A domains arranged in a pseudo-three-fold axis of symmetry. The two C domains are roughly cylindrical and oriented side-by-side to form a pedestal for the A1-A2-A3 rosette. The A3 domain makes extensive contacts with the C1 and A2 domains. The structure also reveals a disulfide bond unique to Ptex-Va, linking Cys642 in the A2 domain with Cys1002 in the A3. These features likely account for the high stability of the molecule even after proteolytic processing of the B domain and/or cleavage between the A1 and A2 domains. Although the C2 domain is significantly more disordered than the other domains, both C1 and C2 each contain protruding loops at their base with hydrophobic residues pointing outward. These structural features replicate those found in inactivated bovine Va and hVIII considered critical for membrane binding by the hemostatic cofactors. Surprisingly, despite the presence of these structures, light scattering measurements revealed negligible binding of Ptex-Va to synthetic membranes composed of phosphatidylcholine and phosphatidylserine with an estimated 103-fold weaker affinity than that of hV. We reasoned that this unexpected property of the venom protein, not conducive to regulated coagulation, was unlikely to be replicated in factor V from the plasma of the snake. Sequence alignment of Ptex-Va with factor V from snake plasma revealed 11 differences in the 328 residues of the C1 and C2 domains. Of these, 9 were located on the distal region of these domains, occupying a band approximately 7 Å thick across the molecule. Mutagenesis of Ptex-Va to introduce these 9 substitutions followed by its expression and purification yielded a derivative that bound to membranes with high affinity in a manner equivalent to hV. This striking gain in function sheds new and unexpected light on the structural determinants of high affinity membrane binding in Ptex-Va and by extension, its homologues hV and hVIII. Our high resolution structure of this hV-like species with a series of unusual properties provides a unique platform to address major but unresolved questions related to the structural correlates of hV function. It also reveals the basis for molecular mimicry whereby a cofactor essential for regulated blood coagulation has served as a scaffold for the evolution of a potent toxin by simultaneous loss in the ability to bind membranes and a gain in the ability to bind its proteinase with high affinity in a membrane-independent fashion. Disclosures:No relevant conflicts of interest to declare.

Active Site-labeled Prothrombin Inhibits Prothrombinase in Vitro and Thrombosis in Vivo

Mouse and human prothrombin (ProT) active site specifically labeled with D-Phe-Pro-Arg-CH(2)Cl (FPR-ProT) inhibited tissue factor-initiated thrombin generation in platelet-rich and platelet-poor mouse and human plasmas. FPR-prethrombin 1 (Pre 1), fragment 1 (F1), fragment 1.2 (F1.2), and FPR-thrombin produced no significant inhibition, demonstrating the requirement for all three ProT domains. Kinetics of inhibition of ProT activation by the inactive ProT(S195A) mutant were compatible with competitive inhibition as an alternate nonproductive substrate, although FPR-ProT deviated from this mechanism, implicating a more complex process. FPR-ProT exhibited ∼10-fold more potent anticoagulant activity compared with ProT(S195A) as a result of conformational changes in the ProT catalytic domain that induce a more proteinase-like conformation upon FPR labeling. Unlike ProT and ProT(S195A), the pathway of FPR-ProT cleavage by prothrombinase was redirected from meizothrombin toward formation of the FPR-prethrombin 2 (Pre 2)·F1.2 inhibitory intermediate. Localization of ProT labeled with Alexa Fluor® 660 tethered through FPR-CH(2)Cl ([AF660]FPR-ProT) during laser-induced thrombus formation in vivo in murine arterioles was examined in real time wide-field and confocal fluorescence microscopy. [AF660]FPR-ProT bound rapidly to the vessel wall at the site of injury, preceding platelet accumulation, and subsequently to the thrombus proximal, but not distal, to the vessel wall. [AF660]FPR-ProT inhibited thrombus growth, whereas [AF660]FPR-Pre 1, lacking the F1 membrane-binding domain did not bind or inhibit. Labeled F1.2 localized similarly to [AF660]FPR-ProT, indicating binding to phosphatidylserine-rich membranes, but did not inhibit thrombosis. The studies provide new insight into the mechanism of ProT activation in vivo and in vitro, and the properties of a unique exosite-directed prothrombinase inhibitor.

A Simplified and High-Throughput Chromogenic Assay for Testing Tissue Factor–Dependent Procoagulant Activity

Tissue factor (TF), the primary initiator of the coagulation cascade, plays a critical role in hemostasis and thrombosis, and inhibition of TF activity appears to be an attractive target for the treatment of cardiovascular diseases. However, few selective small-molecule inhibitors of TF are available, and the present assays for measuring TF activity are relatively expensive and complex. The authors present a simple and high-throughput chromogenic assay for screening TF inhibitors based on using commercial human prothrombin complex instead of purified coagulation factors, reducing the dosage, and performing with a one-stage procedure. In the optimized assay, <45 µL cell lysates was incubated with Tris-CaCl(2) buffer (pH 7.3) containing human prothrombin complex at 37°C for 15 min in 96-well or 384-well plates. Tris-EDTA buffer (pH 8.4) containing chromogenic substrate Xa was then added and the absorbance measured at 405 nm. This simplified assay was more sensitive or precise than some reported methods for TF procoagulant activities. Two known active compounds (curcumin and simvastatin) inhibiting TF activity were tested by the simplified assay to validate the screening method. Furthermore, berberine and cryptotanshinone suppressed TF activity induced by lipopolysaccharides in human monocytes by this assay and might be promising new TF inhibitors.

Interaction of prothrombin with a phospholipid surface: evidence for a membrane-induced conformational change

Prothrombin interacts with phosphatidylserine containing platelet membranes via its N-terminal, γ-carboxyglutamate (gla) residue-rich domain. Once bound it is cleaved to form the active protease, thrombin (factor IIa). Human prothrombin was cleaved with cathepsin G in the absence of calcium and magnesium ions. Under these conditions, the gla domain was removed. Phospholipid protected the protein from this proteolytic event, and this suggests that a conformational change may be induced by interaction with phospholipids. Binding of prothrombin to a surface containing 20% phosphatidylserine/80% phosphatidylcholine was detected by surface plasmon resonance, whereas no interaction with gla-domainless prothrombin was observed. Binding of intact prothrombin in the presence of calcium ions showed complex association kinetics, suggesting multiple modes of initial interaction with the surface. The kinetics of the dissociation phase could be fitted to a two-phase, exponential decay. This implies that there are at least two forms of the protein on the surface one of which dissociates tenfold more slowly than the other. Taken together, these data suggest that, on binding to a membrane surface, prothrombin undergoes a conformational change to a form which binds more tightly to the membrane.

RGD-dependent binding of TP508 to integrin αvβ3 mediates cell adhesion and induction of nitric oxide

TP508, a 23-amino acid RGD-containing synthetic peptide representing residues 508 to 530 of human prothrombin, mitigates the effects of endothelial dysfunction in ischaemic reperfusion injury. The objective of this study was to investigate whether TP508 binds to members of the integrin family of transmembrane receptors leading to nitric oxide synthesis. Immobilised TP508 supported adhesion of endothelial cells and alphavbeta3-expressing human embryonic kidney cells in a dose- and RGD-dependent manner. Soluble TP508 also inhibited cell adhesion to immobilised fibrinogen. The involvement of alphavbeta3 was verified with function-blocking antibodies and surface plasmon resonance studies. Adhesion of the cells to immobilised TP508 resulted in an induction of phosphorylated FAK and ERK1/2. In endothelial cells, TP508 treatment resulted in an induction of nitric oxide that could be inhibited by LM609, an alphavbeta3-specific, function-blocking monoclonal antibody. Finally, TP508 treatment of isolated rat aorta segments enhanced carbachol-induced vasorelaxation. These results suggest that TP508 elicits a potentially therapeutic effect through an RGD-dependent interaction with integrin alphavbeta3.

Prothrombin kringle‐2 induces death of mesencephalic dopaminergic neurons in vivo and in vitro via microglial activation

We have shown that prothrombin kringle-2 (pKr-2), a domain of human prothrombin distinct from thrombin could activate cultured rat brain microglia in vitro. However, little is known whether pKr-2-induced microglial activation could cause neurotoxicity on dopaminergic (DA) neurons in vivo. To address this question, pKr-2 was injected into the rat substantia nigra (SN). Tyrosine hydroxylase (TH) immunohistochemistry experiments demonstrate significant loss of DA neurons seven days after injection of pKr-2. In parallel, pKr-2-activated microglia were detected in the SN with OX-42 and OX-6 immunohistochemistry. Reverse transcription PCR and double-label immunohistochemistry revealed that activated microglia in vivo exhibit early and transient expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and several proinflammatory cytokines. The pKr-2-induced loss of SN DA neurons was partially inhibited by the NOS inhibitor N(G)-nitro-L-arginine methyl ester hydrochloride, and the COX-2 inhibitor DuP-697. Extracellular signal-regulated kinase 1/2, c-Jun N-terminal kinase and p38 mitogen-activated protein kinase were activated in the SN as early as 1 hr after pKr-2 injection, and localized within microglia. Inhibition of these kinases led to attenuation of mRNA expression of iNOS, COX-2 and several proinflammatory cytokines, and rescue of DA neurons in the SN. Intriguingly, following treatment with pKr-2 in vitro, neurotoxicity was detected exclusively in co-cultures of mesencephalic neurons and microglia, but not microglia-free neuron-enriched mesencephalic cultures, indicating that microglia are required for pKr-2 neurotoxicity. Our results strongly suggest that microglia activated by endogenous compound(s), such as pKr-2, are implicated in the DA neuronal cell death in the SN.