Anti-fibrin Antibody Research Articles

Fluorescent peptide for detecting factor XIIIa activity and fibrin in whole blood clots forming under flow

BackgroundDuring clotting, thrombin generates fibrin monomers and activates plasma-derived transglutaminase factor (F) XIIIa; collagen and thrombin-activated platelets offer thrombin-independent cellular FXIIIa (cFXIIIa) for clotting. Detecting fibrin on collagen and tissue factor surfaces in whole blood clotting typically uses complex reagents like fluorescent fibrinogen or antifibrin antibody. ObjectivesWe want to test whether the peptide using the α2- antiplasmin crosslinking mechanism by FXIIIa is a useful tool in both monitoring FXIIIa activity, and visualize and monitor fibrin formation, deposition, and extent of crosslinking within fibrin structures in whole blood clots formed under flow. MethodsWe tested a fluorescent peptide derived from α2-antiplasmin sequence (Ac-GNQEQVSPLTLLKWC-fluorescein) to monitor the location of transglutaminase activity and fibrin during whole blood clotting under microfluidic flow (wall shear rate, 100 s−1). ResultsThe peptide rapidly colocated with accumulating fibrin due to transglutaminase activity, confirmed by Phe-Pro-Arg-chloromethylketone inhibiting fibrin and peptide labeling. The FXIIIa inhibitor T101 had no effect on fibrin generation but ablated the labeling of fibrin by the peptide. Similarly, Gly-Pro-Arg-Pro abated fibrin formation and thus strongly attenuated the peptide signal. At arterial wall shear rate (1000 s−1), less fibrin was formed, and consequently, less peptide labeling of fibrin was detected compared with venous conditions. The addition of tissue plasminogen activator caused a reduction of both fibrin and peptide signals. Also, the peptide strongly colocalized with fibrin (but not platelets) in clots from laser-injured mouse cremaster arterioles. For clotting under flow, FXIIIa activity was most likely plasma-derived since a RhoA inhibitor did not block α2-antiplasmin fragment cross-linking to fibrin. ConclusionUnder flow, the majority of FXIIIa-dependent fibrin labeling with peptide during clotting was distal of thrombin activity. The synthetic peptide provided a strong and sustained labeling of fibrin as it formed under flow.

Agonal Thrombus at Necropsy-A Third Category of Blood Coagulation in Domestic Carnivores.

Agonal thrombus is infrequently discussed in veterinary forensic pathology, being misdiagnosed as postmortem coagulation. The main purpose of the present study is to confirm that agonal thrombosis is an important tool in ruling out sudden death and to characterize it by gross, histological, and immunohistochemical approaches. The investigations have been conducted on 56 domestic carnivores. Fibrin was observed as rosette-like arrangements around platelet aggregates, loose network, wave-like pattern or short fibers and the additional tendency of lines of Zahn being noted inconsistently. All agonal thrombi had positive reactions for anti-CD61 for platelets, disposed in variable-sized clumps or in a linear pattern close to endothelial cells of endocardium. The same positive reaction has been noted to anti-fibrinogen and anti-fibrin antibodies. CD45, CD68 and von Willebrand factor had a very low to absent expression. Cardiac lesions were found in 22 cases (39.29%) suggesting predisposition to agonal thrombosis in animals with cardiovascular diseases. The results prove that agonal thrombus is a third category of blood coagulation that forms strictly during agonal death. The microscopical findings describe the agonal thrombus similar to the morphology of recent thrombus vera. Given the results, the agonal thrombus is a useful tool that confirms the agonal suffering prior to death.

Inhibition of Factor XII Attenuates Prothrombotic Complications in Sickle Cell Mice

Sickle Cell Disease (SCD) is the most common inherited hemoglobinopathy, affecting millions worldwide. Although characterized by chronic hemolytic anemia and recurrent vaso-occlusive episodes, SCD is increasingly recognized as a hypercoagulable state. Indeed, SCD patients have an 11-25% incidence of venous thromboembolism at a median age of 30 years, associated with a 3-fold increased risk of mortality. Moreover, ischemic stroke and silent cerebral infarctions occur in 7-13% of SCD patients. We have previously shown that tissue factor, an initiator of the extrinsic coagulation pathway, contributes to thrombo-inflammation and microvascular cerebral thrombosis in mouse models of SCD . Recently, the intrinsic coagulation pathway, including Factor XII (FXII), has received significant attention because targeting components of this pathway reduces thrombosis without affecting primary hemostasis. We have shown that FXII deficiency reduces plasma markers of thrombin generation and inflammation in sickle mice. However, the contribution of FXII to thrombosis and prothrombotic complications in SCD is not known. In this study we evaluated the effects of blocking FXII activity on venous thrombosis and ischemia/reperfusion (IR)-induced brain injury in SCD mice.First, Townes HbSS mice (SS) and non-sickle Townes HbAA controls (AA) (male and female, 16 weeks) received anti-FXII antibody or control IgGκ1 (10 mg/kg, IV) 30 minutes prior to subjecting them to venous thrombosis, initiated by applying positive current (3 volts, 90 sec) to the femoral vein. To visualize platelet and fibrin accumulation, mice were injected with rhodamine 6G and anti-fibrin antibody 59D8 labeled with Alexa Fluor 647, respectively. The femoral vein thrombi were imaged by intravital fluorescence microscopy using time-lapse capture every 10 seconds, to acquire images of fibrin and platelets over 60 min. The accumulation of platelets and fibrin was quantified for relative intensity of each fluorophore over the region of the observed thrombus. As previously shown, thrombi of SS/IgG mice showed an increased fibrin and platelet accumulation compared to AA/IgG group. Importantly, 15D10 treatment significantly attenuated both fibrin (p<0.001) and platelet (p<0.05) deposition over time in SS mice compared to SS/IgG group. The same effect of 15D10 treatment was observed in AA mice. At the end of experiment, clots were collected and stained with hematoxylin and eosin, and clot volume was assessed histomorphometrically (Nikon Ti-2, FIJI Software). Surprisingly, despite higher fibrin content, clots from SS/IgG mice had significantly smaller volume than clots from AA/IgG group (0.32 ± 0.04 versus 0.60 ± 0.11 mm 3, p<0.05). Importantly, administration of 15D10 significantly reduced clot volume in both SS (0.086 ± 0.01 mm 3, p<0.05) and AA mice (0.1 ± 0.02 mm 3, p<0.05).Next, AA and SS mice (male and female, 8-10 weeks) were subjected to brain IR injury induced by middle cerebral artery occlusion for 60 minutes followed by 24 hours of reperfusion (mouse model of ischemic stroke). 15D10 or control IgGκ1 (10 mg/kg, IV) were injected 30 minutes before occlusion and again at 6 hours into the reperfusion period to generate 3 experimental groups: AA/IgG, SS/IgG and SS/15D10. All analyzed parameters of brain IR injury were significantly worse in the SS/IgG group compared to the AA/IgG group. Compared to IgG, pre-treatment of SS mice with 15D10 significantly attenuated neuronal damage determined by volume of brain infarction (11.7 ± 3.7 vs 24.9 ± 2.4%, p<0.001) and improved behavioral deficit assessed by mean stroke score (9.0 ± 0.9 vs 14.6 ± 0.9, p<0.01). These changes were accompanied by a significant increase in leukocytes rolling (1978.0 ± 93.5 vs 1517.0 ± 180.3 rolling leukocytes/sec/mm 2, p<0.001), and significant reduction in the number of adherent leukocytes (367.2 ± 49.0 vs 723.4 ± 48.5, adherent leukocytes/mm 2, p<0.001) observed in the brain microvasculature of SS mice treated with 15D10 compared to SS/IgG group.Together, our data indicates that in the mouse model of SCD FXII contributes to the experimental venous thrombosis and ischemic stroke. Given that targeting the intrinsic pathway can reduce thrombosis without affecting hemostasis, our data suggest that targeting FXII might be a beneficial treatment in reducing inflammatory and thrombotic complications in SCD patients without a risk of bleeding. DisclosuresWallisch: Aronora Inc,: Current Employment. Key: Grifols: Research Funding; Takeda: Research Funding; BioMarin: Honoraria, Other: Participation as a clinical trial investigator; Sanofi: Consultancy; Uniqure: Consultancy, Other: Participation as a clinical trial investigator. Gruber: Aronora Inc.: Current Employment, Current equity holder in publicly-traded company; Oregon Health and Science University: Current Employment.

Improved Inhibition of Protease-Activated Receptor 4 (PAR4) with Function-Blocking Antibodies Provides Equivalent Antithrombotic Activity in the Face of the Hyper-Reactive Thr120 PAR4 Variant

Background: Thrombin activates platelets via two protease-activated receptors (PARs), PAR1 and PAR4, and both are targets of antithrombotic drugs - a PAR1 inhibitor is approved for clinical use and PAR4 inhibitors are in trial. However, recent studies report a PAR4 variant (rs773902; encoding Thr120 instead of Ala120) is expressed in up to 80% of individuals, depending on the population, and renders the receptor more sensitive to agonists and less sensitive to antagonists. These studies suggest the effectiveness of current strategies to inhibit PAR4 may vary substantially across the population. Here, we report the generation of a novel function-blocking PAR4 antibody that provides equivalent antithrombotic activity against the Ala120 or Thr120 PAR4 variant.Methods: We generated a panel of anti-PAR4 antibodies and examined their inhibitory activity against the Ala120 and Thr120 PAR4 variants in cultured cells, isolated platelets, and in an ex vivo whole blood thrombosis assay.Results: Our lead function-blocking antibody bound human PAR4 with high selectivity (~16-fold over PARs1, 2 or 3) and affinity (KD ~ 5nM). This antibody provided near-complete inhibition of thrombin cleavage of either the Ala120 or Thr120 PAR4 variant when transfected in HEK293T cells. Platelets isolated from individuals expressing the Thr120 PAR4 variant exhibited increased thrombin-induced aggregation and phosphatidylserine exposure versus those expressing the Ala120 PAR4 variant. Yet the anti-PAR4 antibody inhibited these responses with an equivalent IC50 of ~ 5 µg/ml. Furthermore, this function-blocking anti-PAR4 antibody significantly impaired platelet procoagulant activity in an ex vivo whole blood thrombosis assay. Here, equivalent inhibition of thrombin generation (FRET-based thrombin substrate) and fibrin formation (anti-fibrin antibody) was observed in thrombi generated in blood from individuals expressing the Ala120 or Thr120 PAR4 variant.Conclusion: We generated a highly selective and effective function-blocking anti-PAR4 antibody that impairs thrombin-induced PAR4 cleavage, platelet activation, and pro-thrombotic activity equivalently in the presence of the Ala120 or Thr120 PAR4 variant. These findings reveal a novel approach to PAR4 inhibition that overcomes the impaired sensitivity of existing approaches against the commonly expressed Thr120 PAR4 variant, and provide rationale for such an approach for improved antithrombotic therapy. DisclosuresNo relevant conflicts of interest to declare.

In vivo characterization of spontaneous microhemorrhage formation in mice with cerebral amyloid angiopathy

The pathophysiology of microhemorrhages in the context of cerebral amyloid angiopathy (CAA) remains poorly understood. Here we used in vivo two-photon microscopy in aged APP/PS1 mice with mild-to-moderate CAA to assess the formation of microhemorrhages and their spatial relationship with vascular Aβ depositions in the surrounding microvascular network. Mice with chronic cranial windows were intravenously injected with fluorescent dextran to visualize the vessels and a fluorescently labeled anti-fibrin antibody to visualize microhemorrhages. Focal vessel irradiations resulted in extravascular fibrin-positive clots at individual rupture sites that remained visible for weeks. Spontaneous extravascular fibrin-positive clots were more often observed in 19-month-old transgenic APP/PS1 mice compared to their wild-type littermate controls (p = 0.039), after heparin administration. In the transgenic mice, these spontaneous leakage sites frequently occurred at arteriolar segments without CAA at bifurcations or vessel bends. These findings suggest that the presence of vascular Aβ per se does not directly predispose vessels to leak, but that complex flow dynamics within CAA-affected vascular networks likely play a role. Our in vivo approach for the detection of individual spontaneous leakage sites may be used in longitudinal studies aimed to assess structural and functional alterations at the single-vessel level leading up to microhemorrhage formation.

Neutrophil Extracellular Traps and Fibrin in Otitis Media

Bacterial resistance in acute otitis can result in bacterial persistence and biofilm formation, triggering chronic and recurrent infections. To investigate the middle ear inflammatory response to bacterial infection in human and chinchilla temporal bones. Six chinchillas underwent intrabullar inoculations with 0.5 mL of 106 colony-forming units (CFUs) of Streptococcus pneumoniae, serotype 2. Two days later, we counted bacteria in middle ear effusions postmortem. One ear from each chinchilla was processed in paraffin and sectioned at 5 µm. The opposite ear was embedded in epoxy resin, sectioned at a thickness of 1 µm, and stained with toluidine blue. In addition, we examined human temporal bones from 2 deceased donors with clinical histories of otitis media (1 with acute onset otitis media, 1 with recurrent infection). Temporal bones had been previously removed at autopsy, processed, embedded in celloidin, and cut at a thickness of 20 µm. Sections of temporal bones from both chinchillas and humans were stained with hematoxylin-eosin and immunolabeled with antifibrin and antihistone H4 antibodies. Histopatological and imminohistochemical changes owing to otitis media. Bacterial counts in chinchilla middle ear effusions 2 days after inoculation were approximately 2 logs above initial inoculum counts. Both human and chinchilla middle ear effusions contained bacteria embedded in a fibrous matrix. Some fibers in the matrix showed positive staining with antifibrin antibody, others with antihistone H4 antibody. In acute and recurrent otitis media, fibrin and neutrophil extracellular traps (NETs) are part of the host inflammatory response to bacterial infection. In the early stages of otitis media the host defense system uses fibrin to entrap bacteria, and NETs function to eliminate bacteria. In chronic otitis media, fibrin and NETs appear to persist.

Platelet CD36 Potentiates Thrombus Formation in Hyperlipidemic Conditions By Activating Redox Sensitive MAP Kinase ERK5

Atherosclerotic plaque instability is a pathological process that can lead to ischemic emergencies, such as myocardial infarction (MI) and stroke. Thrombosis in this context is promoted by circulating oxidized lipids present in LDL particles (oxLDL), which are generated during plaque formation. These particles are recognized by scavenger receptor CD36 present on platelets. CD36 binding to oxLDL lowers the threshold for platelet activation by recruitment and activation of Src kinases Fyn and Lyn, followed by signaling including generation of reactive oxygen species (ROS) through NADPH oxidase (Nox). Although these effectors are important for the prothrombotic properties of CD36, the downstream signaling that links CD36 to classic agonist-induced platelet activation pathways is incompletely defined. We hypothesize that platelet CD36 promotes thrombosis by generating specific ROS to modulate critical redox-sensitive signaling pathways in hyperlipidemia. Platelet MAP kinase ERK5 is a redox sensor that was shown to be required for optimal platelet activation in MI, a condition with greatly elevated ROS. We previously reported that oxLDL-CD36 signaling leads to the generation of the specific ROS superoxide radical anion (O2●-) and that O2●-/hydrogen peroxide (H2O2) are important mediators of platelet aggregation. Additionally, we reported that platelet ERK5 was activated by oxLDL in a CD36-dependent pathway requiring Src kinases, Nox and O2●-/H2O2 to promote platelet activation. We now report the function of platelet ERK5 in this context by performing an ex vivo microfluidic thrombosis assay in which mice whole blood is perfused over immobilized collagen. Platelet adhesion and accumulation were visualized in real time via mepacrine-tagged platelets. We found that stimulating whole blood from wild type C57Bl6 mice with oxLDL promoted platelet adhesion and accumulation by 12.9% compared to buffer stimulation (p=0.033). Whole blood from CD36 null mice showed indistinguishable platelet adhesion and accumulation when stimulated with oxLDL or buffer, suggesting CD36-dependency. We used the platelet ERK5 null mice, which was generated by crossing ERK5flox mice with PF4-cre+ mice (ERK5flox/PF4-cre+), and showed that platelet adhesion and accumulation by oxLDL was abrogated compared to control ERK5flox mice. Additionally, the in vivo relevance of platelet ERK5 activation by CD36 was determined by performing a novel collagen-mediated murine thrombosis assay. This assay is dependent on syngeneic transplantation of the epigastric artery into the carotid artery, where the collagen-rich outer adventitial layer is exposed to blood flow, thus generating a thrombus. Platelets and fibrin accumulation were visualized in real time by Rhodamine 6G and fluorophore-tagged anti-fibrin antibody, respectively. We transplanted bone marrows from donor ERK5flox mice or ERK5flox/PF4-cre+ mice into recipient atherogenic apoE null mice and generated hyperlipidemia by feeding the mice a high fat diet (HFD). ApoE null mice with ERK5flox bone marrows (apoE:ERK5floxBM) on HFD developed rapid platelet accumulation with subsequent thromboembolisms compared to apoE null mice with platelet ERK5-null bone marrows (apoE:ERK5flox/PF4-cre+BM) (p<0.01 at 10 min), demonstrating that this model is sensitive to detect a prothrombotic phenotype in diet-induced hyperlipidemia. Chow fed mice do not show potentiation of platelet accumulation in both apoE chimeras (p=0.56). Additionally, HFD-fed apoE:ERK5floxBM showed accelerated fibrin accumulation compared to apoE:ERK5flox/PF4-cre+BM, suggesting a role for CD36-ERK5 signaling in platelet procoagulant properties. Control diet-fed apoE null chimeras have indistinguishable fibrin accumulation kinetics. Subsequent studies to determine the mechanism for fibrin accumulation (via fluorophore-tagged Annexin V binding) showed that exposure of platelets to oxLDL induced dose-dependent phosphatidylserine (PS) exposure, a mechanism required for platelets to promote coagulation. Buffer or LDL controls had no effect. A CD36 blocking antibody, FA6, was able to abrogate oxLDL-mediated PS exposure compared to control antibody (p=0.041). These findings suggest that platelet CD36 potentiates thrombosis through a specific redox-regulated pathway requiring ERK5 and that ERK5 is a critical modulator of thrombosis in hyperlipidemic conditions. DisclosuresNo relevant conflicts of interest to declare.

Inhibition of protease‐activated receptor 4 impairs platelet procoagulant activity during thrombus formation in human blood

Essentials The platelet thrombin receptor, PAR4, is an emerging anti-thrombotic drug target. We examined the anti-platelet & anti-thrombotic effects of PAR4 inhibition in human blood. PAR4 inhibition impaired platelet procoagulant activity in isolated cells and during thrombosis. Our study shows PAR4 is required for platelet procoagulant function & thrombosis in human blood. Background Thrombin-induced platelet activation is important for arterial thrombosis. Thrombin activates human platelets predominantly via protease-activated receptor (PAR)1 and PAR4. PAR1 has higher affinity for thrombin, and the first PAR1 antagonist, vorapaxar, was recently approved for use as an antiplatelet agent. However, vorapaxar is contraindicated in a significant number of patients, owing to adverse bleeding events. Consequently, there is renewed interest in the role of platelet PAR4 in the setting of thrombus formation. Objectives To determine the specific antiplatelet effects of inhibiting PAR4 function during thrombus formation in human whole blood. Methods and Results We developed a rabbit polyclonal antibody against the thrombin cleavage site of PAR4, and showed it to be a highly specific inhibitor of PAR4-mediated platelet function. This function-blocking anti-PAR4 antibody was used to probe for PAR4-dependent platelet functions in human isolated platelets in the absence and presence of concomitant PAR1 inhibition. The anti-PAR4 antibody alone was sufficient to abolish the sustained elevation of cytosolic calcium level and consequent phosphatidylserine exposure induced by thrombin, but did not significantly inhibit integrin αII b β3 activation, α-granule secretion, or aggregation. In accord with these in vitro experiments on isolated platelets, selective inhibition of PAR4, but not of PAR1, impaired thrombin activity (fluorescence resonance energy transfer-based thrombin sensor) and fibrin formation (anti-fibrin antibody) in an ex vivo whole blood flow thrombosis assay. Conclusions These findings demonstrate that PAR4 is required for platelet procoagulant function during thrombus formation in human blood, and suggest PAR4 inhibition as a potential target for the prevention of arterial thrombosis.

Abstract 4849: Implications of cancer induced blood coagulation in cancer diagnosis and therapy

Abstract A large body of clinical evidence indicates that abnormal coagulation occurs in a variety of cancers, especially invasive cancers. If cancer clusters erode adjacent normal or tumor vessels, hemorrhage may occur, and fibrin clots immediately form in situ to stop the bleeding. The fibrin clots are subsequently replaced by collagen, similar to the case in the normal wound healing process. Although there are many points in common between cancer-induced stroma and normal wound healing, the critical difference between the two is that the pathophysiological condition in cancer lasts for as long as cancer cells survive in the body (Adv Drug Deliv Rev 2012). First, we successfully developed a mAb that reacts only with human insoluble fibrin, not human fibrinogen (Cancer Sci 2011). In contrast to previously reported anti-fibrin monoclonal antibodies (mAbs), our anti-fibrin clot mAb (clone 102-10) recognizes an unexplored hole that is uncovered only when a fibrin clot forms. The epitope of the 102-10 mAb was mapped to a hydrophobic region on the Bβ-chain that interacted closely with a counterpart region on the γ-chain in the soluble state. New anti-Bβ and anti-γ mAbs specific for peptides lining the discovered hole appeared to bind exclusively to fibrin clots. Analysis of the kinetics of fibrin clot deposition using 102-10 in several disease models indicated that fibrin clot formation occurs only in the acute phase of the disease process, thrombosis, inflammation or trauma, and that these clots virtually disappear within a few weeks to be substituted by collagen. Then, we developed a new PET probe using 102-10. The radiolabelled mAb was injected into mice bearing chemically induced spontaneous tumors, and the probe showed clear and specific accumulation in the tumors. We named this detection strategy cancer stromal targeting (CAST) diagnois. Immunohistochemistry using this mAb revealed higher fibrin deposition in WHO grade 4 gliomas than in lower-grade gliomas. Because erosive tumors are highly likely to show micro-hemorrhages, even early asymptomatic tumors detected with radiolabeled 102-10 mAb may be aggressively malignant (Sci. Rep. 2013). In conclusion, CAST diagnosis and therapy based on anti-insoluble fibrin mAb may be a useful new strategy in the field of oncology. Citation Format: Yohei Hisada, Masahiro Yasunaga, Shingo Hanaoka, Shinji Saijou, Takashi Sugino, Atsushi Tsuji, Tsuneo Saga, Kouhei Tsumoto, Shino Manabe, Jun-ichiro Kuroda, Jun-ichi Kuratsu, Yasuhiro Matsumura. Implications of cancer induced blood coagulation in cancer diagnosis and therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4849. doi:10.1158/1538-7445.AM2014-4849

Severe Acquired Bleeding Tendency Secondary To An Anti-Thrombin Antibody In a Patient With Monoclonal Gammapathy Of Unknown Significance: Direct In Vivo evidence

IntroductionAnti-thrombin antibodies are a rare cause of acquired bleeding disorder. We report a case of a patient with a monoclonal gammapathy of unknown significance (MGUS) referred to us for an acquired bleeding tendency. We found that he had an isolated anti-thrombin antibody and showed that this antibody inhibit platelet accumulation and fibrin generation in vivo in a mouse model of arterial thrombus formation. PatientA 40-year-old man with no personal or familial history of bleeding was referred to us for multiple bleeding episodes (rectal bleeding, hematoma) for 18 years. He was diagnosed 12 years previously with an IgG Kappa MGUS which was stable. Complete blood counts were normal. Activated partial thromboplastin time (APTT) and thrombin time (TT) were prolonged at 76 seconds (control 35) and > 120 seconds (control 18) respectively. The prolonged TT was observed using human and bovine thrombin. A TT mixing study was performed by measuring the TT of serial dilutions of patient's plasma in normal pooled plasma. This study confirmed an acquired anti-thrombin activity of patient's plasma, as TT of the mix was prolonged from dilutions 1:1 to 1:16 (>120 to 25 seconds). Other coagulation factors were normal. No other auto-antibodies were detected except an isolated lupus anticoagulant. Thrombin generation in patient's platelet rich plasma measured using the Calibrated Automated Thrombogram (CAT) showed a prolonged lag time (15.5 versus 4.7 minutes) associated with a decreased endogenous thrombin potential (ETP, 834 versus 1730 nmol/L min). The diagnosis of an acquired anti-thrombin antibody associated with a MGUS was proposed. The patient received multiple treatments, including immunosuppressive agents, anti-CD20 monoclonal antibody therapy and bortezomib, in order to eradicate the antibody with no success. MethodsAnti-thrombin IgG antibodies were affinity purified from the patient's plasma using activated agarose beads covalently coupled to human alpha thrombin (Aminolink, ThermoScientific). A mouse model of thrombosis was used to test the effect of the infusion of the purified anti-thrombin IgG on thrombus formation in vivo. Platelet accumulation and fibrin generation at the site of injury was imaged after laser-induced injury of cremaster arterioles using intravital microscopy in wild type mice. Platelet and fibrin labeling were performed using anti-CD42 antibody conjugated to DyLight 488 (Emfret) and an anti-fibrin antibody conjugated with AlexaFluor 647 (Sekisui). ResultsImmunoblotting using human and bovine alpha-thrombin thrombin confirmed the purified IgG from the patient's plasma (yield 86 µg/ml) to be anti-thrombin antibodies. At a concentration of 2.45 mg/mL, these anti-thrombin IgG were able to prolong APTT and TT (59 versus 33 seconds and >120 versus 19 seconds, respectively). Control IgG (2.5 mg/mL) purified from healthy individuals had no effect on APTT and TT. Using the mouse model of laser-induced arteriolar injury, we found that patient-derived anti-thrombin IgG (690 microg) caused a significant inhibition of platelet accumulation and fibrin generation of more than 70% at the site of vessel wall injury. ConclusionWe report a case of severe acquired bleeding tendency secondary to an anti-thrombin antibody in a patient with an IgG Kappa MGUS. Anti-thrombin IgG antibodies purified from the patient's plasma retain the anti-thrombin activity in vitro. Additionally, infusion of purified anti-thrombin IgG from the patient induced a dramatic decrease in platelet thrombus accumulation and fibrin generation at the site of injury in a mouse model of arterial injury, proving the etiology of patient's bleeding tendency. This is the first description of the anti-thrombotic effect of anti-thrombin antibodies directly in vivo. Disclosures:Turhan:BMS, Novartis: Honoraria, Research Funding.

Thrombodynamics of Microvascular Repairs: Effects of Antithrombotic Therapy on Platelets and Fibrin

To evaluate the hypothesis that platelets and fibrin differentially accrue at microvascular anastomoses in arteries versus veins and under different pharmacologic conditions. We evaluated mouse arterial and venous anastomoses with intravital fluorescence imaging, using fluorophore-labeled platelets and anti-fibrin antibodies to measure the extent of thrombus component development in the intraluminal anastomotic site. We evaluated systemic heparin or eptifibatide (platelet aggregation inhibitor) to determine their relative influences on thrombus composition. Platelets accumulated rapidly in both arterial and venous repairs, and then fell in number after 10 to 30 minutes of reflow. Fibrin had a relatively steady development over 60 minutes in veins, with a more variable increase in arteries. Heparin reduced platelet accumulation in arteries and fibrin development in veins. Eptifibatide reduced platelets in both arteries and veins and had an apparent effect on lowering the amount of fibrin in veins. These findings show that platelets have a rapid, transient response, whereas fibrin has a slower, more sustained accrual in both arterial and venous anastomoses. Furthermore, inhibition of either coagulation or platelet aggregation can influence presumably non-targeted components of thrombosis in vascular repairs of both arteries and veins. Preventing replantation failure using antithrombotic therapies requires a better understanding of the effect of each pharmacologic compound on the various aspects of thrombogenesis.

Abstract 1645: Characterization of a novel anti-insoluble-fibrin chimeric antibody (AFCA) for cancer stromal target (CAST) therapy and diagnosis.

Abstract Background: A large body of clinicopathological evidence supports the conclusion that abnormal coagulation occurs in a variety of cancers. In the non-malignant condition, fibrin clots (insoluble fibrin) form only at the onset or active state of disease and subsequently disappear because of plasmin digestion or replacement with collagen within a few weeks. However, in cancer, fibrin clot formation and subsequent replacement with collagen continue for as long as cancer cells survive in the body. In this context we considered that the fibrin clot could be a pathophysiologically specific target in cancer and accordingly developed an anti-insoluble-fibrin chimeric antibody (AFCA). Here, we characterize AFCA and demonstrate its potential as a tool for drug delivery to the cancer stroma. Method: The specificity of AFCA was evaluated by ELISA using insoluble fibrin, fibrinogen (the precursor of fibrin), soluble fibrin, and D-dimer (the degradation product of insoluble fibrin) as antigens. To compare AFCA with commercially available anti-fibrin antibodies, we performed ELISA with insoluble fibrin, fibrinogen, and D-dimer as antigens. We also conducted western blotting with native fibrinogen and denatured fibrinogen. To evaluate the accumulation of AFCA in tumors, we gave fluorescence-labeled AFCA to transgenic mice with pancreatic cancer. Results: AFCA recognized only insoluble fibrin, not any fibrin-related proteins. AFCA had higher specificity against insoluble fibrin than did commercially available anti-fibrin antibodies. Western blotting analysis revealed that AFCA reacted with denatured fibrinogen but not native fibrinogen. AFCA, but not isotype control IgG, accumulated in the pancreatic cancer tissues. Conclusion: These findings imply that the epitope of AFCA is a distinctive sequence that is uncovered only on the fibrin clot. Because lasting asymptomatic fibrin clot formation is a cancer-specific phenomenon, AFCA should be a useful tool for drug delivery to insoluble fibrin present around cancers. Citation Format: Yohei Hisada, Masahiro Yasunaga, Yasuhiro Matsumura. Characterization of a novel anti-insoluble-fibrin chimeric antibody (AFCA) for cancer stromal target (CAST) therapy and diagnosis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1645. doi:10.1158/1538-7445.AM2013-1645

Complement C3 is a substrate for activated factor XIII that is cross‐linked to fibrin during clot formation

Complement C3 is a substrate for activated factor <scp>XIII</scp> that is cross‐linked to fibrin during clot formation

Reduction of thrombus size in murine models of thrombosis following administration of recombinant α1-proteinase inhibitor mutant proteins

The variant serpin α1-PI M358R inhibits thrombin and other proteases such as activated protein C (APC) and factor XIa. We previously described recombinant proteins HAPI M358R (α1-PI M358R containing an N-terminal extension corresponding to residues 1-75 of heparin cofactor II) and HAPI RCL5 (HAPI M358R with F352-I356 and I360 substituted for the corresponding residues of antithrombin), with enhanced selectivity for thrombin over APC inhibition. We tested the hypotheses that these recombinant proteins would limit thrombosis in three mouse models, and that the HAPI chimeric proteins would be more effective than α1-PI M358R. Recombinant serpins were purified from Escherichia coli by nickel chelate and ion exchange affinity chromatography, and administered to mice intravenously. HAPI RCL5 reduced incorporation of radiolabelled fibrin(ogen) into thrombi in the ferric chloride-injured vena cava in a dose-dependent manner; HAPI M358R was less effective and α1-PI M358R was without effect. In a model of murine endotoxaemia, HAPI RCL5 was more effective than α1-PI M358R in reducing radiolabelled fibrin(ogen) deposition in heart and kidneys; immunohistochemistry of tissue sections showed lesser staining with anti-fibrin(ogen) antibodies with both treatments. In the ferric chloride-injured murine carotid artery, administration of both recombinant serpins was equally effective in lengthening the vessel's time to occlusion. Our results show that the antithrombotic efficacy of the recombinant serpins correlates with their potency as thrombin inhibitors, since HAPI RCL5 inhibits thrombin, but not factors Xa, XIa, XIIa, or neutrophil elastase, more rapidly than α1-PI M358R.

Distinct Roles of the Extrinsic and the Intrinsic Coagulation Pathways In Different Murine Thrombosis Models,

Abstract 3278In vivo vascular lesion models are used to elucidate the pathophysiological mechanisms of thrombosis. We found that the intrinsic and extrinsic coagulation pathways exhibit a distinct relative contribution to arterial occlusion initiated by a FeCl3- or nitrogen dye pulse laser (NDPL)-induced injury evaluated comparatively in the carotid artery (CA), the femoral artery (FA) or the femoral vein (FV). Methods. C57Bl/6J (BL6), IL4Rα/GPIbα-tg (IL4) and VWF−/− mice between 8 and 12 weeks of age were anesthetized with 2% isofluorane, and the right jugular vein was cannulated for IV delivery of fluids. Injury in the exposed left CA was induced by applying a 0.7 ml drop of 7 or 8 % FeCl3 for 3 minutes; variations of blood flow in the injured artery were then monitored with a miniaturized ultrasound flow probe for an additional 30 min. In all experiments, FeCl3 concentrations refer to the hexahydrate form of the salt. The time to first occlusion (flow rate <0.1 ml/min) and the normalized area under the flow rate curve, defined as the Flow Index (FI), were used as endpoints for statistical analysis. In the FA and FV, respectively, vascular injury by FeCl3 was induced by applying a 9% drop for 2.5 minutes or 4% drop for 1 minute; then, platelet and fibrin deposition in forming thrombi were visualized by epifluorescence microscopy of injected green calcein-labeled platelets and AlexaFluor 546-labelled anti-fibrin antibody. The NDPL injury was induced in both FV and FA with two series of 30 s laser light pulses, the second upstream of and 30 s after the first. Images selected at regular time intervals from a 20 min real-time video recording were used for measuring the integrated density of formed thrombi with the ImageJ software. Results. In the CA, 8% FeCl3 reliably caused a stable occlusion that was completely abolished by lepirudin, a thrombin inhibitor, at a calculated 42 μM concentration in blood. Also, IL4 mice, lacking a functional extracellular GPIbα domain, and VWF−/− mice failed to develop a normal thrombus. In C57Bl/6J mice, injection of the anti-mouse factor XI (mFXI) monoclonal antibody (mAB) 14E11 inhibited thrombus formation in a dose dependent manner, and completely when injected at a dose of 0.5 μg/g. In contrast, the anti-mouse tissue factor (mTF) mAB 21E10 had no effect when injected at 25 μg/g; the latter amount of anti-mTF mAB was variably inhibitory only when a presumably milder lesion was induced by a reduced 7% FeCl3 concentration. Of note, however, even at the lower dosage of 9 μg/g the anti-mTF mAB completely prevented CA occlusion when co-injected with an individually non-effective dose (0.065 μg/g) of anti-mFXI mAB. Essentially the same results, albeit obtained with a different methodology, were observed by evaluating thrombus formation in the FA and FV after FeCl3-induced injury. Findings were remarkably different in the NDPL model of FA and FV thrombosis. In this case, platelet aggregate formation was transient and almost completely reversed by 20 minute post-injury, while fibrin deposition at the site of injury increased progressively in time. Fibrin deposition was essentially absent in the IL4 mouse but, contrary to what we observed in the same vascular beds after FeCl3 injury, it was only partially inhibited by injecting the anti-mFXI mAB at dosage as high as 1 μg/g. Moreover, and again in contrast with the results after FeCl3 injury, the anti-mTF mAB injected at 25 μg/g by itself significantly inhibited fibrin deposition. Conclusions. Our findings indicate that distinct thrombogenic vascular lesions elicit a varied response by the extrinsic and intrinsic coagulation pathways, as reflected by the different effects of inhibiting initiating events mediated by TF or FXI activation by FXIIa (the reaction blocked by mAB 14E11), respectively. Moreover, and again in a lesion-specific manner, the two coagulation pathways exhibit a variably relevant synergistic function in leading to thrombin generation as evidenced by fibrin deposition. These results, and the underlying mechanisms presently under investigation, suggest that the thrombogenic potential of different pathological lesions in human vascular disease is also likely varied and may be influenced by lesion-specific characteristics. Consequently, more direct mechanistic evidence must be generated to establish how a given animal thrombosis model may reflect the pathogenesis of acute vascular occlusion in human disease. Disclosures:Gruber:Aronora, LLC: Consultancy, Equity Ownership.

A Fibrin-Specific Thrombolytic Nanomedicine Approach to Acute Ischemic Stroke

To develop a fibrin-specific urokinase nanomedicine thrombolytic agent. In vitro fibrin-clot dissolution studies were utilized to develop and characterize simultaneous coupling and loading of anti-fibrin monoclonal antibody and urokinase onto perfluorocarbon nanoparticle (NP) surface. In vivo pharmacokinetics and fibrin-specific targeting of the nanolytic agent was studied in dogs. Simultaneous coupling of up to 40 anti-fibrin antibodies and 400 urokinase enzymes per perfluorocarbon NP produced an effective targeted nanolytic agent with no significant surface protein-protein interference. Fibrin clot dissolution was not improved by increasing homing capacity from 10 to 40 antibodies/NP, but increasing enzymatic payload from 100 to 400/NP resulted in maximized lytic effect. Fluorescent microscopy showed that rhodamine-labeled urokinase nanoparticles densely decorated the intraluminal thrombus in canine clots in vivo analogous to the fibrin pattern, while an irrelevant-targeted agent had negligible binding. This agent offers a vascularly constrained, simple to administer, low-dose nanomedicine approach that may present an attractive alternative for treating acute stroke victims.

MPO-ANCA-Positive Anti-glomerular Basement Membrane Antibody Disease Successfully Treated by Plasma Exchange and Immunosuppressive Therapy

Anti-glomerular basement membrane (GBM) antibody disease is clinically manifested as rapidly progressive glomerulonephritis (RPGN) with crescentic changes. The renal prognosis is poor. We report here the case of a 61-year-old woman with myeloperoxidase antineutrophil cytoplasmic antibody (MPO-ANCA)-positive anti-GBM antibody disease. This patient was referred to our hospital because of RPGN. Anti-GBM antibody was positive with a titer of 38 EU. The MPO-ANCA titer was 65 EU. Chest imaging examination revealed pulmonary multiple nodules. ANCA-associated vasculitis was suspected. Renal pathology revealed cellular crescents in 13 out of 17 glomeruli. Immunofluorescence with anti-IgG antibody, anti-C3 antibody, and anti-fibrin antibody showed linear staining along the glomerular capillary walls. Based on these findings, the patient was diagnosed with anti-GBM antibody disease. Hemodialysis was started because of uremic syndrome with elevated serum creatinine (6.84 mg/dL). In addition, treatment with plasma exchange using 3.6 L (90 mL/kg) of fresh frozen plasma combined with an oral dose of 40 mg of prednisolone was initiated. Within 3 weeks, both types of autoantibodies became undetectable. Subsequently, this patient achieved dialysis independence and remission of glomerulonephritis. No adverse effects were observed. In patients with MPO-ANCA-positive anti-GBM antibody disease, intensive therapy predominantly with plasma exchange might be operative, even though renal function is less likely to recover.

Abstract 5580: Nanolytic Approach to Prompt Revascularization in Acute Ischemic Stroke

Background: Vascular-constrained fibrin-specific nanolytic agents could permit early revascularization in the setting of acute ischemic stroke. Development of fibrin-targeted nanolytics requires optimized surface co-presentation of homing ligands and fibrinolytic enzymes on nanoparticles without compromising bioactivity. Objective : The study optimized human urokinase coupling and anti-fibrin homing ligand density for targeted thrombolytic efficacy and demonstrated the concept in vivo. Methods and Results: Minimal thiolation (N-succinimidyl-S-acetylthioacetate, SATA) of urokinase (i.e., 1 to 3 SATA/molecule) diminished human clot dissolution time relative to the native molecule; greater degrees of thiolation (3 to 6 SATA/molecule) severely decreased activity. For dog clots, which digested slower than human clots, 1 to 3 SATA/urokinase did not further impair fibrinolysis rate. Concomitant coupling of thiolated urokinase and anti-fibrin antibody (50:5, 100:10, 200:20, and 400:40 ratio, respectively) to perfluorocarbon nanoparticles resulted in optimal clot dissolution for the 100:10 and 200:20 formulations. Increasing the surface urokinase-to-antibody ratio from 0:10 to 50:10, 100:10, 200:10, and 400:10 increased particle size from 273 nm (0:10) to 340 nm at the 50:10 and 100:10 enzyme levels, and to more than 400 nm with 200 urokinase molecules per particle. Acoustic microscopy (25MHz) was used to quantify binding of the fibrin-targeted PFC nanoparticles to acoustically transparent human clots. Fibrin targeting with 10 antibodies per particle provided minimal acoustic enhancement in comparison with the relatively increased (p&lt;0.05) but equivalent signals obtained with 20 or 40 antibodies per particle. Dissolution of canine femoral clots, created by electrical injury, was monitored with low MI ultrasound under open circulation conditions following in situ targeting with the nanolytic system. Substantial subjective clot dissolution and fragmentation was observed in three animals over 60 minutes, demonstrating the nanolytic concept. Conclusion: Nanolytic agents can target fibrinolytic enzymes to lyse nascent thrombus, which may permit early revascularization in acute ischemic stroke with minimal hemorrhagic risk.

Design of biomedical nanodevices for dissolution of blood clots

Currently, tissue plasminogen activator (tPA) is administered intravenously after myocardial infarction and stroke to ensure blood-clot dissolution. One problem associated with such treatment is systemic toxicity of tPA, which can act upon plasminogen both in circulation and at the clot site, generating systemic plasmin and rendering the patient highly vulnerable to hemorrhaging. Here we report the synthesis and in vitro characterization of prototype nanodevices capable of binding to fibrin clots and initiating their dissolution. The devices consist of tPA and antifibrin antibody covalently attached to a 40 nm polystyrene-latex nanoparticles. These nanoscale devices can directly deliver tPA to the clot site through fibrin-specific antibody. In vitro fibrinolysis assay showed that protein–nanoparticle conjugates were only slightly less potent than free tPA. Moreover, tPA activity in the absence of clots was more than 3-fold less than that of the free enzyme. This property can be critical for therapeutic applications because nanoparticles can be expected to dissolve clots at approximately the same rate as free tPA while simultaneously cleaving much less plasminogen in the circulation, lowering the risk of hemorrhage. This system could therefore become a promising agent for thrombolytic treatment.

Endogenous Arteriolar Fibrinolysis Requires Urokinase Plasminogen Activator and Leukocyte Urokinase Plasminogen Activator Receptor-1.

Background: In venous thrombi, urokinase associated with leukocytes activates plasminogen to initiate endogenous fibrinolysis. Leukocytes also interact with arteriolar thrombi.Objectives: We hypothesized that urokinase plasminogen activator (uPA) activity would also be delivered to arteriolar thrombi by leukocytes.Methods: Using established techniques of high-speed intravital fluorescence microscopy to observe the cremaster muscle of mice, we measured platelet and fibrin accumulation in thrombi generated by a laser injury to arterioles. The accumulation of platelets in a thrombus is quantitated by measuring the fluorescence from anti-CD41 antibody tagged with a fluorescent secondary antibody and the presence of fibrin is detected using a fluorescence-tagged anti-fibrin antibody (T2G1).Results: In C57BL/6 wild type mice, fibrin increased to a maximum at about three minutes after thrombus formation and then decreased, such that by eight minutes after thrombus formation fibrin is present at 34% of its maximal value. When wild type mice are pretreated with epsilon-aminocaproic acid (16 mg/kg IV), the decrease in thrombus fibrin content was less (to 54% of maximal 8 minutes after thrombus formation), implying that the fibrin loss is at least partially the result of plasmin activity. Thrombus fibrin accumulation in tissue-plasminogen activator deficient mice (Plat) was greater, peaking at 229% the amount found in wild type mice, but the amount of fibrin present at eight minutes after thrombus formation was not dissimilar from wild type mice (45% ± 5% v. 34% ± 3%, P=0.09). In uPA-deficient mice (Plau) and uPA-receptor-1-deficient mice (Plaur), the amount of maximal thrombus fibrin was 189% and 273% that of fibrin content in wild type mice. Also, although thrombus fibrin did decrease over time in Plau and Plaur mice, it decreased to only 51% and 48% of maximal (each were P < 0.005 v. wild type) at eight minutes after thrombus formation. When wild type leukocytes, isolated from blood by differential centrifugation and sucrose density gradient, were transfused into Plaur mice, thrombus fibrin loss was restored to wild type levels (38% of maximal at 8 minutes after thrombus formation, P=0.36 vs. wild type, P=0.016 vs. Plaur), implying that transfused wild type leukocytes, which express uPA-receptor-1, can rescue fibrinolysis in uPA-receptor-1-deficient mice.Conclusions: These results confirm that endogenous fibrinolytic activity in vivo occurs soon after arteriolar thrombus formation. The plasminogen activators that are required for this fibrinolysis to occur are uPA, and the uPA-receptor-1 on leukocytes. These findings are compatible with the hypothesis that leukocytes can deliver plasminogen activator activity to arteriolar thrombi to initiate endogenous fibrinolysis.