Fibrinogen Γ-chain Research Articles

NanoGraphene Clot: A New Fibrinogen-Mimic Hemostatic Material.

Trauma is the leading cause of death for adults under the age of 44. Internal bleeding remains a significant challenge in medical emergencies, necessitating the development of effective hemostatic materials that could be administered by paramedics before a patient is in the hospital and treated by surgeons. In this study, we introduce a graphene oxide (GO)-based PEGylated synthetic hemostatic nanomaterial with an average size of 211 ± 83 nm designed to target internal bleeding by mimicking the role of fibrinogen. Functionalization of GO-g-PEG with peptides derived from the α-chain of fibrinogen, such as GRGDS, or the γ-chain of fibrinogen, such as HHLGGAKQAGDV:H12, was achieved with peptide loadings of 72 ± 6 and 68 ± 15 μM, respectively. In vitro studies with platelet-rich plasma (PRP) under confinement demonstrated aggregation enhancement of 39 and 24% for GO-g-PEG-GRGDS and GO-g-PEG-H12, respectively, compared to buffer, while adenosine diphosphate (ADP) alone induced a 5% aggregation. Compared to the same materials in the absence of ADP, GO-g-PEG-GRGDS achieved a 47% aggregation enhancement, while GO-g-PEG-H12 a 25% enhancement. This is particularly important for injectable hemostats and highlights the fact that our nanographene-based materials can only act as hemostats in the presence of agonists, reducing the possibility of unwanted clotting during circulation. Further studies on collagen-coated wells under dynamic flow revealed statistically significant augmentation of PRP fluorescence signal using GRGDS- or H12-coated GO-g-PEG compared to controls. Hemolysis studies showed <1% lysis of red blood cells (RBCs) at the highest PEGylated nanographene concentration. Finally, whole human blood coagulation studies reveal faster and more pronounced clotting using our nanohemostats vs PBS control from 3 min and below (blood is clotted with 10% CaCl2 within 4-5 min), with the biggest differences to be shown at 2 and 1 min. At 1 min, the clot weight was found to be ∼45% of that between 4 and 5 min, while no clot was formed in PBS-treated blood. Reduction of CaCl2 to 5 and 3%, or utilization of prostaglandin E1, an anticoagulant, still leads to clots but of smaller weight. The findings highlight the potential of our fibrinogen-mimic PEGylated nanographene as a promising non-hemolytic injectable scaffold for targeting internal bleeding, offering insights into its platelet aggregation capabilities under confinement and under dynamic flow as well as its pronounced coagulation abilities.

Early treatment with Fibrinogen γ-chain peptide-coated, ADP-encapsulated Liposomes (H12-(ADP)-liposomes) ameliorates post-partum hemorrhage with coagulopathy caused by amniotic fluid embolism in rabbits

Amniotic fluid embolism is an unpredictable and sometimes lethal complication of childbirth. Fibrinogen γ-chain peptide-coated, ADP-encapsulated Liposomes (H12-(ADP)-liposomes), which were developed as a platelet substitute, may be useful to control postpartum hemorrhage with consumptive coagulopathy. This study aimed to establish a hemodynamically stable amniotic fluid embolism animal model and evaluate the efficacy of H12-ADP-liposome infusion in the initial management of postpartum hemorrhage complicated with amniotic fluid embolism-involved coagulopathy. Pregnant New Zealand white rabbits (28th day of pregnancy or normal gestation period of 29-35 days) underwent cesarean delivery, followed by intravenous administration of amniotic fluid (a total of 3.0 mL administered in 4 doses over 9 minutes). Thereafter, uncontrolled postpartum hemorrhage was induced by transecting the right midartery and concomitant vein in the myometrium. After initial bleeding for 5 minutes, rabbits received isovolemic fluid resuscitation through the femoral vein with an equivalent volume of blood loss every 5 minutes for 60 minutes. The transfusion regimens included platelet-rich plasma, platelet-poor plasma, and a bolus administration of H12-ADP-liposomes followed by platelet-poor plasma transfusion (8 rabbits per group). Moreover, 60 minutes after initiation of bleeding, rabbits received surgical hemostasis by ligation of bleeding vessels, except in cases with spontaneous hemostasis. The administration of amniotic fluid caused thrombocytopenia (56±3 × 103/μL) and prolonged both clotting time (before administration: 130.0±3.0 to 171.0±5.0 seconds) and prothrombin time (4.5±0.1 to 4.7±0.1 seconds). After the initial 5-minute bleeding in the rabbits, the mean arterial pressure fell to 43±2 mm Hg. Platelet-poor plasma transfusion alone further prolonged clotting time and prothrombin time at 60 minutes (192.0±10.0 and 5.2±0.1 seconds, respectively) with decreasing mean arterial pressure to <40 mm Hg. By contrast, the administration of H12-ADP-liposomes followed by platelet-poor plasma transfusion reduced the prolonged clotting time (153.0±5.0 seconds) and prothrombin time (4.9±0.1 seconds) similar to platelet-rich plasma transfusion (154.0±11.0 and 4.9±0.1 seconds, respectively) at 60 minutes. These rabbits maintained a mean arterial pressure of >45 mm Hg throughout the experiment. H12-ADP-liposome infusion and platelet-poor plasma transfusion and platelet-rich plasma transfusion yielded spontaneous hemostasis in 4 of 8 rabbits, whereas platelet-poor plasma transfusion did not stop bleeding in any of the rabbits. The total blood loss was 59±17 mL in the H12-ADP-liposomes and platelet-poor plasma group, which was half of that in the platelet-poor plasma group (124±10 mL). H12-ADP-liposome infusion may be effective in the initial management of postpartum hemorrhage complicated with amniotic fluid embolism, resulting in mitigation of consumptive coagulopathy.

TRANSMISSION ELECTRON MICROSCOPY FOR THE DIRECT ANALYSIS OF FIBRIN CLOT STRUCTURE

The aim of our study was to compare the structure of clots formed as a result of thrombin-induced fibrin polymerization in the presence or absence of monoclonal fibrin-specific antibodies fragments as factors that change the clot structure. We concentrated on the final stage of fibrin clot formation at maximal turbidity point for every sample. Methods. Fibrin polymerization was studied by transmission electron microscopy (TEM) of negatively contrasted samples on H-600 Transmission Electron Microscope (“Hitachi”,Japan); 1% water solution of uranyl acetate (“Merck”, Germany) was used as a negative contrast. For sample preparation, in sterile glass tubes were sequentially added 0.32 mg/mL human fibrinogen, 0.025 M CaCl2 in 0.05 M ammonium formiate buffer (pH 7.9), and a total sample volume was 0.22 mL. The polymerization of fibrin was initiated by the introduction of thrombin at a final concentration of 0.25 NIH/mL. After 180 s, aliquots were taken from the polymerization medium. Each aliquot was diluted to a final fibrinogen concentration of 0.07 mg/mL; 0.01 mL probes of fibrinogen solution were transferred to a carbon lattice, which was treated with a 1% uranyl acetate solution after 2 minutes. Investigations were per-formed using an H-600 electron microscope at 75 kV. Electron microscopic images were obtained at magnification of 20,000 -50,000. Results. Two monoclonal antibodies fragments were obtained towards the mixture of separated Aα-, Bβ- and γ-chains of fibrinogen. Antibodies fragments that were marked as III-1D and I-4A, had different epitopes within fragment Аα105-206 of D-region of fibrinogen. It was shown that addition of antibody fragment I-4A lead to formation of abnormal fibrils that were thinner than in the control sample and were organized in the dense network (Figure). Control sample exhibited the thick fibrils with well-structured classically organized network. The difference between control and I-4A samples demonstrated that antibody I-4A disrupted the structure of polymerized fibrin. In the same time the fibrils obtained in the presence of antibody fragment III-1D were closer to the control ones. Conclusions. TEM is an informative method for the study of the fibrin network formation. Its application allows to estimate the disruption in fi brin formation directly. In a combination with turbidity study and other functional tests TEM can provide important information about molecular mechanisms of clot formation.

Altered fibrinogen γ-chain cross-linking in mutant fibrinogen-γΔ5 mice drives acute liver injury

BackgroundHepatic deposition of cross-linked fibrin(ogen) occurs alongside platelet accumulation as a hallmark of acetaminophen (APAP)-induced liver injury. ObjectivesWe sought to define the precise role of the fibrinogen γ-chain C-terminal integrin αIIbβ3 binding domain in APAP-induced liver injury. MethodsMice expressing mutant fibrinogen incapable of engaging integrin αIIbβ3 due to a C-terminal fibrinogen γ-chain truncation (mutant fibrinogen-γΔ5 [FibγΔ5] mice) and wild-type mice were challenged with APAP (300 mg/kg, intraperitoneally). ResultsWe observed an altered pattern of fibrin(ogen) deposition in the livers of APAP-challenged FibγΔ5 mice. This led to the unexpected discovery that fibrinogen γ-chain cross-linking was altered in the livers of APAP-challenged FibγΔ5 mice compared with that in wild-type mice, including absence of γ-γ dimer and accumulation of larger molecular weight cross-linked γ-chain complexes. This finding was not unique to the injured liver because activation of coagulation did not produce γ-γ dimer in plasma from FibγΔ5 mice or purified FibγΔ5 fibrinogen. Sanger sequencing predicted that the fibrinogen-γΔ5 γ-polypeptide would terminate at lysine residue 406, but liquid chromatography tandem mass spectrometry analysis revealed that this critical lysine residue was absent in purified fibrinogen-γΔ5 protein. Interestingly, hepatic deposition of this uniquely aberrantly cross-linked fibrin(ogen) in FibγΔ5 mice was associated with exacerbated hepatic injury, an effect not recapitulated by pharmacologic inhibition of integrin αIIbβ3. ConclusionThe results indicate that fibrinogen-γΔ5 lacks critical residues essential to form γ-γ dimer in response to thrombin and suggest that hepatic accumulation of abnormally cross-linked fibrin(ogen) can exacerbate hepatic injury.

Discovery of Fibrinogen γ-chain as a potential urinary biomarker for renal interstitial fibrosis in IgA nephropathy

BackgroundIgA nephropathy (IgAN) is a major cause of chronic kidney disease (CKD). Renal interstitial fibrosis is a hallmark of CKD progression. Non-invasive biomarkers are needed to dynamically evaluate renal fibrosis. Data independent acquisition (DIA)-based liquid chromatography-mass spectrometry (DIA-MS) was used to identify candidate urinary biomarkers in IgAN patients with different renal interstitial fibrosis degrees.MethodsEighteen biopsy-proven IgAN patients and six healthy controls were recruited in a discovery cohort. Interstitial fibrosis changes were evaluated according to Oxford MEST-C scores. Urinary samples were analyzed with DIA-MS to identify hub proteins. Hub proteins were then confirmed by enzyme-linked immunosorbent assay (ELISA) in a validation cohort and the associated gene mRNA expression was analyzed using public gene expression omnibus (GEO) datasets.ResultsComplement and coagulation cascades pathway was the main KEGG pathway related to the over-expressed proteins. Fibrinogen γ-Chain (FGG) was selected as the potential urinary marker for further validation. Urinary FGG to creatinine ratio (uFGG/Cr) levels were higher in both disease controls and IgAN group than in healthy controls, but were not significantly different between IgAN and disease groups. uFGG/Cr was confirmed to be increased with the extent of renal fibrosis and presented moderate correlations with T score (r = 0.614, p < 0.01) and eGFR (r = -0.682, p < 0.01), and a mild correlation with UTP (r = 0.497, p < 0.01) in IgAN group. In disease control group, uFGG/Cr was higher in patients with T1 + 2 compared to those with T0. uFGG/Cr had a good discriminatory power to distinguish different fibrosis stages in IgAN: interstitial fibrosis ≤ 5% (minimal fibrosis) vs. interstitial fibrosis (mild fibrosis) > 5%, AUC 0.743; T0 vs. T1 + 2, AUC 0.839; T0 + 1 vs. T2, AUC 0.854. In disease control group, uFGG/Cr showed better performance of AUC than UTP between minimal and mild fibrosis (p = 0.038 for Delong’s test). Moreover, GSE104954 dataset showed that FGG mRNA expression was up-regulated (fold change 1.20, p = 0.009) in tubulointerstitium of IgAN patients when compared to healthy living kidney donors.ConclusionUrinary FGG is associated with renal interstitial fibrosis and could be used as a noninvasive biomarker for renal fibrosis in IgAN.

Production, extraction and characterization of a serine protease with fibrinolytic, fibrinogenolytic and thrombolytic activity obtained by Paenibacillus graminis

This study provides optimized parameters for producing and purifying a microbial serine protease by Paenibacillus graminis in submerged fermentation and the innovative use of endosperm of Gliricidia sepium as substrate. Biphasic aqueous systems were the method of choice for the partial purification of this protease. After extracting the enzyme, its biochemical characterization and fibrinolytic, fibrinogenolytic and thrombolytic activity were performed. The highest protease activity (16.25 U/mL) was obtained in the PEG-rich phase in the composing assay of MPEG (8000 g/mol), CPEG (12.5 g/mol), CPHO (10%) and pH 8.0 (optimum pH). Recoveries greater than 80% were obtained with a purification factor of 5.21, a partition coefficient of 1.13 and a yield of 145.52%. The fibrinolytic protease (50 kDa) purified achieved a 46.96% reduction of thrombus in vitro and managed to fully degrade the Bβ-chain of human fibrinogen and the γ-chain of bovine fibrinogen. The enzyme activity was enhanced by Cu2+, Zn2+, Na+, Ca2+ and K+. These results suggest that the protease purified by ATPS can potentially be used as a future agent thrombolytic.

A novel fibrinogen γ-chain frameshift mutation, p. Cys365Phefs*41, causing hypofibrinogenemia with bleeding phenotype in a Chinese family.

BackgroundCongenital hypofibrinogenemia is a rare bleeding disease that is classified as the quantitative deficient type. In the present study, investigated the relationship between the genotype and phenotype in a family with hypofibrinogenemia.MethodsThe proband was aware of a predisposition to bleeding. Functional analysis was performed for her all family members, including coagulation function tests, thrombus molecular markers, thromboelastography, scanning electron microscopy, DNA sequencing, and high-performance liquid chromatography-mass spectrometry (HPLC-MS). Pathogenicity analysis and protein modeling of mutant amino acids were also performed.ResultsA novel heterozygous mutation in c.1094delG was detected in FGG exon 8, which resulted in p. Cys365Phefs*41 (containing the signal peptide) in the proband and her mother, who showed a corresponding decrease in fibrinogen function and levels. Thromboelastography indicated that the strength of their blood clots decreased and they had an increased risk of bleeding. The proband fibrin network structure was looser than healthy controls, with large pores in the network, which increased the permeability of lytic enzymes. Results of HPLC-MS showed a lack of mutant peptide chain expression in their plasma, indicating that the family had congenital hypofibrinogenemia, with a clinical phenotype that is related to the degree of fibrinogen deficiency. The mutation truncated the γ-peptide chain and destroyed the functional structure of fibrinogen, including the γ352Cys-γ365Cys disulfide bond. The truncated peptide chains may also lead to nonsense-mediated decay.ConclusionsThe mutation induced a structural change at the carboxyl-terminal of the fibrinogen molecule, leading to fibrinogen secretion dysfunction.

Association of fibrinogen and plasmin inhibitor, but not coagulation factor XIII gene polymorphisms with coronary artery disease.

BackgroundIn the final phase of clot formation, fibrinogen constitutes frame, whereas factor XIII (FXIII) active form is responsible for the covalent cross-linking of fibrin fibres and plasmin inhibitor (PI), thus contributing to clot stability. It could be expected that any change of coagulation factors' structure affects the clot formation and modulates the atherothrombotic risk. The aim was to determine the frequency of four single nucleotide polymorphisms: (i) A > G in codon 312 of the fibrinogen α-chain gene (rs6050, Thr312AlaFGA), (ii) C > T at position 10034 of the 3 - untranslated region in the fibrinogen γ-chain gene (rs2066865, 10034C > T FGG), (iii) C > T in codon 564 of the FXIII-A subunit gene (rs5982, Pro564LeuFXIII-A), and (iv) C > T in codon 6 of the plasmin inhibitor gene (rs2070863, Arg6TrpPI) in Croatian patients and their association with coronary artery disease (CAD).MethodsWe performed the unrelated case-control association study on the consecutive sample of patients 18 years old, who had undergone coronary angiography for investigation of chest pain and suspected CAD. The cases were patients with confirmed CAD (N=201), and the controls were the subjects with no CAD (N=119). Samples were genotyped using PCR-RFLP analysis.ResultsObserved frequencies of the rare alleles of Thr312Ala FGA, 10034C > T FGG, Leu564Pro FXIII-A and Arg6Trp PI polymorphisms were 21%, 17%, 14%, 20%, respectively. Patients with 10034C > T FGG CC genotype had 3.5 times (95% CI 1.02-12.03) higher adjusted odds for CAD than patients with 10034C > T FGG TT genotype. Patients with Arg6Trp PI CC genotype had 3.86 times (95% CI 1.23-12.12) higher odds for CAD than patients with Arg6Trp PI TT genotype. It seems that those genotype-related higher odds are also male-gender related. No difference was observed regarding any other investigated polymorphism.ConclusionsOur finding suggests that 10034C > T FGG and Arg6Trp PI are associated with CAD.

Focal adhesion kinase and Src mediate microvascular hyperpermeability caused by fibrinogen- γC- terminal fragments.

ObjectivesWe previously reported microvascular leakage resulting from fibrinogen-γ chain C-terminal products (γC) occurred via a RhoA-dependent mechanism. The objective of this study was to further elucidate the signaling mechanism by which γC induces endothelial hyperpermeability. Since it is known that γC binds and activates endothelial αvβ3, a transmembrane integrin receptor involved in intracellular signaling mediated by the tyrosine kinases FAK and Src, we hypothesized that γC alters endothelial barrier function by activating the FAK-Src pathway leading to junction dissociation and RhoA driven cytoskeletal stress-fiber formation.Methods and resultsUsing intravital microscopy of rat mesenteric microvessels, we show increased extravasation of plasma protein (albumin) resulting from γC administration. In addition, capillary fluid filtration coefficient (Kfc) indicated γC-induced elevated lung vascular permeability. Furthermore, γC decreased transendothelial barrier resistance in a time-dependent and dose-related fashion in cultured rat lung microvascular endothelial cells (RLMVECs), accompanied by increased FAK/Src phosphorylation detection by western blot. Experiments with pharmacological inhibition or gene silencing of FAK showed significantly reduced γC-induced albumin and fluid leakage across microvessels, stress-fiber formation, VE-cadherin tyrosine phosphorylation, and improved γC-induced endothelial barrier dysfunction, indicating the involvement of FAK in γC mediated hyperpermeability. Comparable results were found when Src was targeted in a similar manner, however inhibition of FAK prevented Src activation, suggesting that FAK is upstream of Src in γC-mediated hyperpermeability. In addition, γC-induced cytoskeletal stress-fiber formation was attenuated during inhibition or silencing of these tyrosine kinases, concomitantly with RhoA inhibition.ConclusionThe FAK-Src pathway contributes to γC-induced microvascular barrier dysfunction, junction protein phosphorylation and disorganization in a manner that involves RhoA and stress-fiber formation.

Heterozygous variant fibrinogen γA289V (Kanazawa III) was confirmed as hypodysfibrinogenemia by plasma and recombinant fibrinogens.

Congenital fibrinogen disorders are classified as afibrinogenemia, hypofibrinogenemia, dysfibrinogenemia, and hypodysfibrinogenemia. However, difficulties are associated with discriminating between dysfibrinogenemia, hypofibrinogenemia, and hypodysfibrinogenemia using routine analyses. We previously reported a heterozygous variant fibrinogen (γA289V; Kanazawa III) as hypodysfibrinogenemia; however, the same variant had previously been described as hypofibrinogenemia. To clarify the production of γA289V fibrinogen, we expressed recombinant γA289V (r-γA289V) fibrinogen and compared it with wild-type (WT) and adjacent recombinant variant fibrinogens. Target mutations were introduced into a fibrinogen γ-chain expression vector by site-directed mutagenesis, and the vector was then transfected into Chinese hamster ovary cells to produce recombinant fibrinogen. Fibrinogen was purified from the plasma of the proposita, and culture media and fibrinogen functions were analyzed using fibrin polymerization, plasmin protection, and FXIIIa-catalyzed fibrinogen cross-linking. The fibrinogen concentration ratio of the culture media to cell lysates was markedly lower for r-γA289V fibrinogen than for WT. Because the secretion of recombinant γF290L (r-γF290L) fibrinogen was similar to WT, we compared r-γF290L fibrinogen functions with WT. The fibrin polymerization of Kanazawa III plasma (K-III) fibrinogen was significantly weaker than normal plasma fibrinogen. Moreover, K-III fibrinogen showed a markedly reduced "D:D" interaction. However, all functions of r-γF290L fibrinogen were similar to WT. An in silico analysis confirmed the above results. The present results demonstrated that γA289 is crucial for the γ-module structure, and the γA289V substitution markedly reduced fibrinogen secretion. Moreover, K-III fibrinogen showed markedly reduced fibrin polymerization and "D:D" interactions. γA289V fibrinogen was confirmed as hypodysfibrinogenemia.

Purification and Assays of Tachylectin-5.

Tachylectin-5, a 41-kDa protein with a common fold of the C-terminal globular domain of the γ-chain of fibrinogen, is purified from horseshoe crab hemolymph plasma by affinity column chromatography, using acetyl-group-immobilized resin. Two types of isolectins, tachylectin-5A and tachylectin-5B, are obtained by stepwise elution with GlcNAc at 25 and 250mM, respectively. Tachylectins-5A and -5B exhibit extraordinarily strong hemagglutinating activity against all types of human erythrocytes (the minimum agglutinating concentration of 0.004-0.008μg/mL for tachylectin-5A and 0.077-0.27μg/mL for tachylectin-5B). Their hemagglutinating activities are inhibited by acetyl group-containing sugars and noncarbohydrates such as sodium acetate, acetylcholine, and acetyl CoA (the minimum inhibitory concentrations of 1.3-1.6mM), indicating that the acetyl group is required and sufficient for recognition by tachylectins-5A and -5B. EDTA inhibits their hemagglutinating activity, whereas the inhibition is overcome by adding an excess amount of Ca2+. Tachylectins-5A and -5B also exhibit bacterial agglutinating activity against both Gram-negative bacteria (the minimum agglutinating concentrations of 0.04-0.08μg/mL for tachylectin-5A and 0.05-0.11μg/mL for tachylectin-5B) and Gram-positive bacteria (the minimum agglutinating concentrations of 0.3-2.4μg/mL for tachylectin-5A and 15.1-26.8μg/mL for tachylectin-5B). Interestingly, tachylectins-5A and -5B enhance the antimicrobial activity of a hemocyte-derived peptide, big defensin.

Dominant Role of αIIbβ3 in Platelet Interactions with Polymerizing Cross-Linked Fibrin

The formation of a stable platelet thrombus in vivo is accompanied by the generation of thrombin and the conversion of fibrinogen to cross-linked fibrin. This requires thrombin's action on fibrinogen, producing fibrin monomers, and factor XIII, catalyzing the reciprocal transamidation of the C-terminal γ-chain peptides from adjacent fibrinogen molecules. While the interaction of platelets with fibrinogen has been shown to be mediated by the interaction of fibrinogen γ-chain residues 404-411 with the RGD pocket on integrin αIIbβ3, the interaction with cross-linked fibrin is less well understood despite its clinical relevance. We previously reported that activated, but not unactivated, αIIbβ3 can support binding to D-dimer, the cross-linked plasmin fragment of fibrin, and 'D98' a fibrinogen fragment lacking the γ404-411 sequence. Here we studied the interaction of washed platelets in buffer containing 1 mM Mg2+ with polymerizing fibrin in an aggregometer by adding 0.2 U/ml thrombin and monitoring light transmission. To prevent the interaction of γ12 with the RGD pocket, studies were performed in the presence of 150 μM RGDW, a concentration that completely eliminates aggregation of the platelets to a thrombin receptor activating peptide (T6). RGDW eliminated the initial wave of platelet aggregation induced by thrombin, but did not prevent the appearance of a delayed wave (DW), which appeared after ~4-5 min (Panel A-B). Parallel immunoblot studies demonstrated that cross-linking of fibrinogen γ-chains, (γ-γ dimers) appeared within 1 min after adding thrombin and was ~85% complete by 5 min (Panel C). Thus, both cross-linked and uncross-linked fibrin(ogen) was present during the DW. Adding fibrinogen at 10-100 μg/ml enhanced the amplitude of the DW in a dose-dependent manner (Panel D). The fibrin polymerization inhibitor Gly-Pro-Arg-Pro (GPRP; 5 mM) dramatically inhibited the amplitude of the DW, supporting the importance of fibrin polymerization (Panel B). An antibody to platelet receptor α2β1 (6F1) had no effect on the slope or amplitude of the DW, whereas an antibody to GPIb (6D1) had a mild effect on the slope but not the amplitude of the DW. Since GPVI has variably been reported to interact with fibrin, we tested whether there was evidence of GPVI-mediated signaling during the DW. The known GPVI ligands/activators collagen and collagen-related peptide (CRP) (controls), produced marked increases 3 min after the onset of aggregation in phosphorylation of PLC-γ2-Y759, Syk-Y525/526, and LAT-Y191. Thrombin alone caused a major increase in phosphorylation of Syk-Y525/526 and a slight increase in phosphorylation of PLCγ2-Y759; however, adding RGDW dramatically inhibited the phosphorylation of Syk. We conclude that at 3 min after the onset of the DW there is minimal or no GPVI-mediated signal transduction. Similarly, neither the Src inhibitor PP2 at 100 μM or the Syk inhibitor VI at 1 μM, both of which inhibit CRP-induced platelet aggregation, inhibited the DW. Since polymerizing fibrin has the potential for multiple interactions with different platelet αIIbβ3 receptors, thus increasing the avidity, we compared the ability of αIIbβ3 antagonists to inhibit T6-induced aggregation versus the DW of polymerizing fibrin. Whereas the γ400-411 peptide was able to nearly completely inhibit T6-induced platelet aggregation at ~150 μM, it had virtually no impact on the DW at concentrations up to 500 μM. RGDW eliminated T6-induced aggregation at ~5 μM, but required 100-fold as much to produce ~80% inhibition of the DW. Similarly, eptifibatide was able to nearly eliminate T6-induced aggregation at ~5 μM, but 100-fold as much was required to eliminate the DW. Thus, even if αIIbβ3 antagonists do not inhibit the DW at doses that eliminate T6-induced aggregation, one cannot infer that other receptors are involved. Consistent with a dominant role for activated αIIbβ3 in the process, 10 mM EDTA essentially eliminated the DW and reptilase-induced fibrin polymerization did not lead to platelet incorporation unless the platelets were activated with T6 or another αIIbβ3 activator. In addition, both D-dimer and D98 inhibited the thrombin-induced DW at 200 µg/ml. We conclude that activated αIIbβ3 plays a dominant role in supporting high avidity platelet interactions with polymerizing, partially cross-linked, fibrin, and that the mechanism has features akin to, but distinct from the interaction of αIIbβ3 with fibrinogen. Figure Disclosures Coller: CeleCor: Consultancy, Equity Ownership, Research Funding; Scholar Rock: Consultancy, Equity Ownership; Centocor/Janssen: Patents &amp; Royalties: abxicimab; Accumetrics/Instrumentation Laboratory: Patents &amp; Royalties: VerifyNow assay.

Plasma fibrin clot proteomics in healthy subjects: Relation to clot permeability and lysis time

BackgroundLittle is known about fibrin clot composition in relation to its structure and lysability. We investigated plasma clots protein composition and its associations with clot properties. MethodsWe studied 20 healthy subjects aged 31–49 years in whom plasma fibrin clot permeability (Ks) and clot lysis time (CLT) were determined. A proteomic analysis of plasma fibrin clots was based on quantitative liquid chromatography-mass spectrometry. ResultsAmong 494 clot-bound proteins identified in all clots, the highest concentrations were for fibrinogen chains (about 64% of the clot mass) and fibronectin (13%). α2-antiplasmin (2.7%), factor XIIIA (1.2%), complement component C3 (1.2%), and histidine-rich glycoprotein (HRG, 0.61%) were present at relatively high concentrations. Proteins present in concentrations <0.5% included (pro)thrombin, plasminogen, apolipoproteins, or platelet factor 4 (PF4). Fibrinogen-α and -γ chains were associated with age, while body-mass index with clot-bound apolipoproteins (all p < .05). Ks correlated with fibrinogen-γ and PF4 amounts within plasma clots. CLT was associated with fibrinogen-α and -γ, PF4, and HRG (all p < .05). ConclusionsThis study is the first to show associations of two key measures of clot properties with protein content within plasma clots, suggesting that looser fibrin clots with enhanced lysability contain less fibrinogen-γ chain, platelet-derived PF4, and HRG. SignificanceOur study for the first time suggests that more permeable fibrin clots with enhanced lysability contain less fibrinogen-γ chain, platelet-derived factor 4, and histidine-rich glycoprotein, which is related to accelerated clot lysis. The current findings might have functional consequences regarding clot structure, stability, and propagation of thrombin generation, and detailed proteomic analysis of clots in various disorders opens new perspective for coagulation and fibrin research.

Combination therapy using fibrinogen γ-chain peptide-coated, ADP-encapsulated liposomes and hemoglobin vesicles for trauma-induced massive hemorrhage in thrombocytopenic rabbits.

We previously developed substitutes for red blood cells (RBCs) and platelets (PLTs) for transfusion. These substitutes included hemoglobin vesicles (HbVs) and fibrinogen γ-chain (dodecapeptide HHLGGAKQAGDV, H12)-coated, adenosine diphosphate (ADP)-encapsulated liposomes [H12-(ADP)-liposomes]. Here, we examined the efficacy of combination therapy using these substitutes instead of RBC and PLT transfusion in a rabbit model with trauma-induced massive hemorrhage with coagulopathy. Thrombocytopenia (PLT count approximately 40,000/μL) was induced in rabbits by repeated blood withdrawal and isovolemic transfusion with autologous RBCs. Thereafter, lethal hemorrhage was induced in rabbits by noncompressible penetrating liver injury. Subsequently, H12-(ADP)-liposomes with platelet-poor plasma (PPP), platelet-rich plasma (PRP), or PPP alone were administered to stop bleeding. Once achieving hemostasis, HbVs, allogenic RBCs, or 5% albumin were transfused into rabbits to rescue them from fatal anemia following massive hemorrhage. Administration of H12-(ADP)-liposomes/PPP as well as PRP (but not PPP) effectively stopped liver bleeding (100% hemostasis). The subsequent administration with HbVs as well as RBCs after hemostasis markedly rescued rabbits from fatal anemia (75% and 70% survivals for 24 hr, respectively). In contrast, 5% albumin administration rescued none of the rabbits. Combination therapy with H12-(ADP)-liposomes and HbVs may be effective for damage control resuscitation of trauma-induced massive hemorrhage.

Diminished Renal Pathology in a Mouse Model of Sickle Cell Anemia in Which Fibrinogen Binding to Mac-1 Is Inhibited

Sickle cell anemia (SCA) is caused by a point mutation in the beta-globin gene. SCA has potentially devastating consequences including chronic hemolytic anemia, episodic vascular occlusion, inflammation and oxidative stress, and cumulative multi-organ damage resulting in early mortality. In fact, with reduction of childhood mortality due to early diagnosis and infection prophylaxis, end-organ damage is the major cause of death in SCA. SCA patients show hyper coagulative state in the absence of vascular occlusion. Recently, our group has shown that reduction of circulating major clotting factor, thrombin, in SCA mice significantly improves survival, reduces inflammation, results in reduced multi-organ damage and prolongs survival (Arumugam 2015). However, the mechanism(s) by which thrombin contributes to the pathophysiology of SCA remains undefined. While fibrinogen functions primarily to occlude blood vessels after cleavage by thrombin into fibrin, and thereby prevents excessive bleeding, it also plays an important role in the pathologic inflammatory disease processes through mitogenic, chemotactic, and immune-regulatory activities by interacting with neutrophils/ macrophages via the Mac-1 receptor. To test the hypothesis that leukocyte engagement of fibrinogen via Mac-1 and secondary inflammation are drivers of SCA-associated organ pathologies, hematopoietic stem cells (HSC) from the well-characterized humanized murine model of SCA, Berkeley sickle mice (HbS or SS) were transplanted into the Fibγ390-396A mice, the later expressing a mutant fibrinogen γ-chain which does not interact with Mac-1 (named as F1M SS). Fibγ390-396 mutation has no effect on clotting functions, supports platelet adhesion, and does not cause spontaneous hemorrhagic events in mice during development or adulthood (Flick 2004). As a control, normal fibrinogen expressing mice were also transplanted with HSC from sickle mice (named as F1WT SS). One year after HSC transplant, the F1M SS and F1WT SS mice were analyzed for blood parameters, reactive oxygen species (ROS), inflammatory cytokines, and organ functions and pathologies. We found that genetically imposed inhibition of fibrinogen binding to Mac-1 resulted in mild reduction (not statistically significant) of neutrophil, monocyte, and platelet counts. There was no significant difference in RBC parameters between F1WT SS and F1M SS mice. However, we found an impressive reduction in WBC ROS and decreased circulating inflammatory cytokines, IL-6 (79.1 ± 19.5 vs. 303.5 ± 73.9, P < 0.001), KC (132.7 ± 19.4 vs. 200.7 ± 33.6, P=0.04) and TNF-α (25.9 ± 2.8 vs. 37.7 ± 3.9, P=0.02) in F1M SS mice compared to F1WT SS mice. We also found significant improvement in SCA-associated glomerular pathologies such as reduced glomerulosclerosis, inflammatory cell infiltration, ischemic lesions, mesangial thickening, mesangial hyper cellularity and glomerular enlargement; associated with reduced albuminuria (123.3 ± 19.5 vs. 322.1 ± 91.4, P=0.03) in F1M SS mice compared to F1WT SS mice. However, tubular pathologies and urine osmolality were not improved in F1M SS mice. RNA-Seq analyses of glomerular preparation revealed improved glomerular protective responses and diminished loss of podocyte/mesangial cell signatures in F1M SS mice compared to F1WT SS mice. Interestingly, unlike the pan-organ improvement with thrombin reduction (Arumugam, 2015), fibrinogen binding to Mac-1 had minimal to no effect on cardiac, lung, and liver functions and pathologies. Taken together, our data support that fibrinogen significantly contributes to the WBC-driven inflammation and ROS production, and represents a key driver of SCA-associated glomerulopathy, and may provide a novel therapeutic target against irreversible kidney damage in SCA. DisclosuresMullins:Shire: Honoraria, Membership on an entity's Board of Directors or advisory committees. Malik:CSL Behring: Patents & Royalties.

Assessing the osteogenic potential of zirconia and titanium surfaces with an advanced in vitro model

ObjectivesIn recent years, zirconia dental implants have gained increased attention especially for patients with thin gingival biotypes or patients seeking metal-free restoration. While physical and chemical material surface properties govern the blood-material interaction and subsequent osseointegration processes, the organizational principles underlying the interplay of biochemical and biophysical cues are still not well understood. Therefore, this study investigated how the interaction of a microstructured zirconia surface with blood influences its osseointegration potential compared to microstructured titanium with or without additional nanostructures. MethodsMicrostructured zirconia and micro- (and nano)structured titanium surfaces were fabricated via sandblasting followed by acid etching and their topographical as well as physico-chemical features were thoroughly characterized. Following, an advanced in vitro approach mimicking the initial blood interaction of material surfaces upon implantation was applied. Fibrinogen adsorption, human blood coagulation as well as their influence on cell fate decisions of primary human bone and progenitor cells (HBC) were studied. ResultsObtained surface micro- and nanostructures on titanium surfaces were sharp with rugged peaks whereas zirconia surfaces were less rough with structures being shallower, more round and granular. Compared to titanium surfaces, the zirconia surface showed increased fibrinogen adsorption, higher levels of total accessible fibrinogen γ-chain moieties yielding in increased platelet adhesion and activation and consequently thrombogenicity. Mineralization of HBC on microstructured surfaces was significantly higher on zirconia than on titanium, but was significantly lower compared to titanium surfaces with nanostructures. SignificanceThis study provides insights into blood-material interaction and subsequent cellular events that are important for implant surface development.

Congenital hypodysfibrinogenemia associated with a novel deletion of three residues (γAla289_Asp291del) in fibrinogen.

Hypodysfibrinogenemia is the least frequently reported congenital fibrinogen disorder, characterized by both quantity and quality defects of fibrinogen. In this study, we investigated the molecular basis of hypodysfibrinogenemia in a Chinese family. Functional fibrinogen was measured by Clauss method, and the antigenic fibrinogen was measured by immunoturbidimetry assay. All the exons and exon-intron boundaries of fibrinogen genes (FGA, FGB and FGG) were analysed by direct DNA sequencing. To further evaluate its molecular and functional characterizations, fibrinogen was purified from the plasma of propositus, then SDS-PAGE, fibrin polymerization, clot lysis, and electron microscopy scanning were all performed. The propositus showed a slight decrease of immunologic fibrinogen (1.52g/L) but dramatically reduced functional fibrinogen (0.3g/L). DNA sequencing revealed a novel heterozygous CCTTTGATG deletion in the exon 8 of FGG, leading to the deletion of Ala289, Phe290, and Asp291 in fibrinogen γ-chain. The polymerization of the fibrinogen from the propositus was markedly impaired, with prolonged lag period and decreased final turbidity. The fibrinogen clottability showed a reduced fraction of participating clot formation. While the clot lysis showed normal. Scanning electron microscopy revealed that the fibers of the propositus were thicker than normal, with larger pores and curlier meshworks. We conclude that γAla289_Asp291del is responsible for the hypodysfibrinogenemia in this case.

Proteomic analysis of plasma from children with sickle cell anemia and silent cerebral infarction.

Silent cerebral infarction is the most common neurological abnormality in children with sickle cell anemia, affecting 30-40% of 14 year olds. There are no known biomarkers to identify children with silent cerebral infarcts, and the pathological basis is also unknown. We used an unbiased proteomic discovery approach to identify plasma proteins differing in concentration between children with and without silent cerebral infarcts. Clinical parameters and plasma samples were analysed from 51 children (mean age 11.8 years, range 6-18) with sickle cell anemia (HbSS). A total of 19 children had silent cerebral infarcts and 32 normal MRI; the children with silent infarcts had lower HbF levels (8.6 vs. 16.1%, P=0.049) and higher systolic blood pressures (115 vs. 108.6, P=0.027). Plasma proteomic analysis showed 13 proteins increased more than 1.3 fold in the SCI patients, including proteins involved in hypercoagulability (α2-antiplasmin, fibrinogen−γ chain, thrombospondin-4), inflammation (α2-macroglobulin, complement C1s and C3), and atherosclerosis (apolipoprotein B-100). Higher levels of gelsolin and retinol-binding protein 4 were also found in the population with silent infarcts, both of which have been linked to stroke. We investigated the genetic basis of these differences by studying 359 adults with sickle cell disease (199 with silent cerebral infarcts, 160 normal MRIs), who had previously undergone a genome-wide genotyping array. None of the genes coding for the differentially expressed proteins were significantly associated with silent infarction. Our study suggests that silent cerebral infarcts in sickle cell anemia may be associated with higher systolic blood pressure, lower HbF levels, hypercoagulability, inflammation and atherosclerotic lipoproteins.

Congenital hypofibrinogenemia associated with γK232T

SummaryWe have previously reported a case of congenital hypofibrinogenemia caused by a novel heterozygous A→C transition at nucleotide 5864 of FGG, leading to the K232T substitution in the fibrinogen γ-chain. However, the pathogenic mechanism is still unclear. To further reveal its molecular basis, we examined the effects of γK232T substitution on fibrinogen synthesis, stability, and secretion through in vitro expression of mutant γ232T in Chinese hamster ovary (CHO) cells. Quantitative RT-PCR of the variant γ-chain mRNA showed that the γ232T transcribed the variant cDNA. Enzyme-linked immunosorbent assay and Western blot analysis of the cell lysates and culture media showed that the CHO cells transfected with successfully synthesized the variant fibrinogen, but failed to secrete it into the culture medium. Furthermore, fibrinogen purified from the plasma of patient showed a normal thrombin-catalyzed fibrin polymerization, also indicating the impeding secretion of variant γ232T fibrinogen. In conclusion, our data reveal that the γK232T is responsible for the congenital hypofibrinogenaemia through interfering with the correct secretion of fibrinogen.

A Novel Thrombolytic and Anticoagulant Serine Protease from Polychaeta, Diopatra sugokai.

Ischemic stroke can result from blockage of blood vessels, forming fibrin clots in the body and causing irreparable brain damage. Remedial thrombolytic agents or anticoagulants have been studied; however, because the FDA-approved tissue plasminogen activator has low efficacy and side effects, it is necessary to develop safer and more effective treatment candidates. This study aimed at assessing the fibrinolytic and anticoagulation features of a novel serine protease extracted and purified from Diopatra sugokai, a polychaeta that inhabits tidal flats. The purified serine protease was obtained through ammonium sulfate precipitation, affinity chromatography, and ion-exchange chromatography. Its molecular size was identified via SDS-PAGE. To characterize its enzymatic activities, the protease activity at various pH and temperatures, and in the presence of various inhibitors, was measured via azocasein assay. Its fibrinolytic activity and anticoagulant effect were assessed by fibrin zymography, fibrin plate assay, and fibrinogenolytic activity assays. The novel 38 kDa serine protease had strong indirect thrombolytic activity rather than direct activity over broad pH (4-10) and temperature (37°C-70°C) ranges. In addition, the novel serine protease exhibited anticoagulant activity by degrading the α-, β-, and γ-chains of fibrinogen. In addition, it did not produce cytotoxicity in endothelial cells. Therefore, this newly isolated serine protease is worthy of further investigation as a novel alkaline serine protease for thrombolytic therapy against brain ischemia.