Thrombin Activity Research Articles

Endothelial membrane mimetic coating modified dialyzer for hemocompatible and anticoagulant-free hemodialysis

Current hemodialysis requires a dilemmatic balance between under- and over-heparinization to prevent extracorporeal circuit clotting and bleeding complications. Here we report an endothelial membrane mimetic coating (EMMC) strategy to replace the systemic heparinization by enhancing the hemocompatibility and anticoagulation property of all the blood contacting surfaces. The EMMC is created by precoating of polydopamine, multi-point anchoring of a sulfobetaine and carboxyl side-chain copolymer (PSBC) and grafting of unfractionated heparin (UFH). The multi-point anchored PSBC layer forms an ultra-thin cell outer membrane zwitterion mimicking interface to enhance hemocompatibility, while the grafted UFH molecules mimic anticoagulant molecules attached on endothelial membrane to endow superior anticoagulation activity. Compared to a commercial polysulfone (PSf) dialysis membrane, the EMMC coated membrane can reduce protein adsorption, platelet adhesion and complement activation by 94.1 %, 98.9 % and 77 %, respectively. Meanwhile, anticoagulation properties, including clotting time, surface thrombus formation time and thrombin inactivation efficacy, are significantly improved compared to currently reported heparin coatings. More importantly, anticoagulant-free extracorporeal circulation using the EMMC modified PSf dialyzer circuit in a beagle dog model was performed smoothly for 2 h without any observable thrombus. Furthermore, main dialysis parameters of the modified hemodialyzer were not decreased. In conclusion, this highly hemocompatible and strong anticlotting endothelial membrane mimetic coating has great potential to replace the systemic heparinization in hemodialysis.

Thrombin activation of the factor XI dimer is a multistaged process for each subunit

BackgroundFactor (F)XI can be activated by proteases, including thrombin and FXIIa. The interactions of these enzymes with FXI are transient in nature and therefore difficult to study. ObjectivesTo identify the binding interface between thrombin and FXI and understand the dynamics underlying FXI activation. MethodsCrosslinking mass spectrometry was used to localize the binding interface of thrombin on FXI. Molecular dynamics simulations were applied to investigate conformational changes enabling thrombin-mediated FXI activation after binding. The proposed trajectory of activation was examined with nanobody 1C10, which was previously shown to inhibit thrombin-mediated activation of FXI. ResultsWe identified a binding interface of thrombin located on the light chain of FXI involving residue Pro520. After this initial interaction, FXI undergoes conformational changes driven by binding of thrombin to the apple 1 domain in a secondary step to allow migration toward the FXI cleavage site. The 1C10 binding site on the apple 1 domain supports this proposed trajectory of thrombin. We validated the results with known mutation sites on FXI. As Pro520 is conserved in prekallikrein (PK), we hypothesized and showed that thrombin can bind PK, even though it cannot activate PK. ConclusionOur investigations show that the activation of FXI is a multistaged procedure. Thrombin first binds to Pro520 in FXI; thereafter, it migrates toward the activation site by engaging the apple 1 domain. This detailed analysis of the interaction between thrombin and FXI paves a way for future interventions for bleeding or thrombosis.

The effects and mechanisms of modified Xiaoyaosan on chronic unpredictable mild stress (CUMS)-induced depressive mice based on network pharmacology

Ethnopharmacological relevanceClinical research and basic scientific experiments have shown that modified Xiaoyaosan (MXYS) has antidepressant effects, whose system mechanism however has not been thoroughly characterized. Aim of the studyThis research was aimed at evaluating the treatment effects of MXYS on chronic unpredictable mild stress (CUMS)-induced depressive mice and exploring underlying mechanisms. Materials and methodsWhether MXYS has effects on depression was investigated via the depressive behaviors of mice, electron microscopy, real-time quantitative polymerase chain reaction (RT-qPCR), Western blot analysis, immunofluorescence (IF) staining and the stereotaxic injection of adeno-associated viruses (AAVs). In addition, network pharmacology was applied to predict relevant molecular targets and possible mechanisms and perform further in vivo validation. ResultsMXYS is effective in ameliorating the depression-like symptoms of CUMS mice. It can stimulate autophagosome formation, activate the expression of microtubule-associated protein 1 light chain 3 (LC3B), autophagy-related gene 5 (Atg5), Atg7 and neuron-specific nuclear protein (NeuN), and decrease the protein expression sequestosome 1 (SQSTM1/p62). The autophagy-upregulating effect of MXYS was weakened by silencing. The network pharmacology analysis revealed that mitogen-activated protein kinase 1 (MAPK1), MAPK3, serine/threonine-protein kinase (AKT1), proto-oncogene tyrosine-protein kinase (SRC), PI 3 kinase p85 alpha (PIK3R1), catenin (cadherin-associated protein) beta 1 (CTNNB1) and human thrombin activator 1 (HRAS) may be of importance to treat depression by MXYS. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that metabolic and autophagy pathways, pathways in cancer and MAPK, phosphoinositide 3-kinase (PI3K)-Akt and rhoptry-associated protein 1 (Rap1) signaling pathways are involved in the antidepressant effects of MXYS. As suggested by Western blot, the anti-depression mechanism of MXYS is possibly associated with the extracellular signal-regulated protein kinase (ERK)/P38 MAPK signaling pathway. ConclusionThe findings indicate the possible antidepressant effects of MXYS on CUMS mice via triggering autophagy to alleviate neuronal apoptosis and prompting autophagy, which may involve the ERK/P38 MAPK signaling pathway.

Detection of Prethrombotic State Malignant Neoplasms, Including Pancreatic Cancer, Using Clot Waveform Analysis

Background: Cancer, especially pancreatic cancer, is frequently associated with thrombosis which is one of the causes of poor outcomes; moreover hypercoagulability can be present in cancer patients. Hypercoagulability is considered to be caused by a thrombin burst. Methods: Activated Partial Tthromboplastin Time (APTT), small amount of tissue factor induced FIX activation assay (sTF/FIXa) and Thrombin Time (TT) assessment using Clot Waveform Analysis (CWA) were performed in 138 patients with malignant neoplasms, including pancreatic cancer. Results: The first derivative peak (1st DP) time (1st DPT), 1st DP height (1st DPH) and 1st DPH/1st DPT ratio were increased in a clotting-factor-FVIII-dependent manner. Thrombosis was frequently associated with pancreatic cancer and was observed in the early stage. CWA-APTT and CWA-sTF/FIXa indicated that the peak times and heights were markedly longer and higher, respectively, in cancer patients, especially pancreas cancer patients, than in patients without cancer. The 1st DPH/1st DPT ratios of CWA-sTF/FIXa were significantly high in patients with pancreas cancer (median value 1.5). CWA-TT showed that the peak times were significantly shorter in cancer patients than in healthy volunteers and that the peak heights were significantly higher in cancer than in benign pancreas diseases. The cutoff value of the 1st DPH/ 1st DPT of sTF/FIXa for cancer patients with thrombosis vs. all patients without cancer was 1.3. Conclusions: Cancer patients, including those with pancreatic cancer were frequently associated with thrombosis due to hypercoagulability caused by thrombin burst detected by CWA. A high 1st DPH/1st DPT ratio of sTF/FIXa may suggest an association with cancer or thrombosis.

Discovering peptide inhibitors of thrombin as a strategy for anticoagulation.

Unusual blood clots can cause serious health problems, such as lung embolism, stroke, and heart attack. Inhibiting thrombin activity was adopted as an effective strategy for preventing blood clots. In this study, we explored computational-based method for designing peptide inhibitors of human thrombin therapeutic peptides to prevent platelet aggregation. The random peptides and their 3-dimentional structures were generated to build a virtual peptide library. The generated peptides were docked into the binding pocket of human thrombin. The designed strong binding peptides were aligned with the native binder by comparative study, and we showed the top 5 peptide binders display strong binding affinity against human thrombin. The 5 peptides were synthesized and validated their inhibitory activity. Our result showed the 5-mer peptide AEGYA, EVVNQ, and FASRW with inhibitory activity against thrombin, range from 0.53 to 4.35 μM. In vitro anti-platelet aggregation assay was carried out, suggesting the 3 peptides can inhibit the platelet aggregation induced by thrombin. This study showed computer-aided peptide inhibitor design can be a robust method for finding potential binders for thrombin, which provided solutions for anticoagulation.

Identification of Factor XIII β-Sandwich Residues Mediating Glutamine Substrate Binding and Activation Peptide Cleavage.

Factor XIII (FXIII) forms covalent crosslinks across plasma and cellular substrates and has roles in hemostasis, wound healing, and bone metabolism. FXIII activity is implicated in venous thromboembolism (VTE) and is a target for developing pharmaceuticals, which requires understanding FXIII - substrate interactions. Previous studies proposed the β-sandwich domain of the FXIII A subunit (FXIII-A) exhibits substrate recognition sites. Recombinant FXIII-A proteins (WT, K156E, F157L, R158Q/E, R171Q, and R174E) were generated to identify FXIII-A residues mediating substrate recognition. Proteolytic (FXIII-A*) and non-proteolytic (FXIII-A°) forms were analyzed for activation and crosslinking activities toward physiological substrates using SDS-PAGE and MALDI-TOF MS. All FXIII-A* variants displayed reduced crosslinking abilities compared to WT for Fbg αC (233 - 425), fibrin, and actin. FXIII-A* WT activity was greater than A°, suggesting the binding site is more exposed in FXIII-A*. With Fbg αC (233 - 425), FXIII-A* variants R158Q/E, R171Q, and R174E exhibited decreased activities approaching those of FXIII-A°. However, with a peptide substrate, FXIII-A* WT and variants showed similar crosslinking suggesting the recognition site is distant from the catalytic site. Surprisingly, FXIII-A R158E and R171Q displayed slower thrombin activation than WT, potentially due to loss of crucial H-bonding with neighboring activation peptide (AP) residues. In conclusion, FXIII-A residues K156, F157, R158, R171, and R174 are part of a binding site for physiological substrates [fibrin (α and γ) and actin]. Moreover, R158 and R171 control AP cleavage during thrombin activation. These investigations provide new molecular details on FXIII - substrate interactions that control crosslinking abilities.

Argatroban promotes recovery of spinal cord injury by inhibiting the PAR1/JAK2/STAT3 signaling pathway.

Argatroban is a synthetic thrombin inhibitor approved by U.S. Food and Drug Administration for the treatment of thrombosis. However, whether it plays a role in the repair of spinal cord injury is unknown. In this study, we established a rat model of T10 moderate spinal cord injury using an NYU Impactor Moder III and performed intraperitoneal injection of argatroban for 3 consecutive days. Our results showed that argatroban effectively promoted neurological function recovery after spinal cord injury and decreased thrombin expression and activity in the local injured spinal cord. RNA sequencing transcriptomic analysis revealed that the differentially expressed genes in the argatroban-treated group were enriched in the JAK2/STAT3 pathway, which is involved in astrogliosis and glial scar formation. Western blotting and immunofluorescence results showed that argatroban downregulated the expression of the thrombin receptor PAR1 in the injured spinal cord and the JAK2/STAT3 signal pathway. Argatroban also inhibited the activation and proliferation of astrocytes and reduced glial scar formation in the spinal cord. Taken together, these findings suggest that argatroban may inhibit astrogliosis by inhibiting the thrombin-mediated PAR1/JAK2/STAT3 signal pathway, thereby promoting the recovery of neurological function after spinal cord injury.

Structure-based virtual screening towards the discovery of novel thrombin inhibitors with Anti-HCC activities.

Hepatic carcinoma (HCC) is one of the most lethal malignant tumors in the world, and new treatment regimens for this disease are urgently needed. Studies have shown that thrombin stimulates tumor progression by forming fibrin and activating platelets. Dabigatran etexilate, a thrombin inhibitor, can inhibit the activity of thrombin and prevent the proliferation and metastasis of HCC in cells and nude mice. The present study was designed to find thrombin inhibitors with novel skeletons, and further confirm the correlation between thrombin inhibition and HCC prevention to identify potential anti-HCC drug leads. The potential thrombin inhibitors were firstly screened in the Topscience Database, and 20 potential active molecules were found by molecular docking. The effect of these molecules on thrombin inhibition, coagulation and tumor proliferation were evaluated, and the definite activity of ZXX-4 was identified. Further in vivo assays in nude mice showed that ZXX-4 inhibited tumor proliferation in nude mice, reduced tumor metastasis, and enhanced the clinical efficacy of first-line drug sorafenib for the treatment of HCC. ZXX-4 can be further explored as an anti-tumor lead compound with a novel skeleton, and inhibition of thrombin can serve as a potential treatment strategy for HCC.

Design and synthesis of dabigatran etexilate derivatives with inhibiting thrombin activity for hepatocellular carcinoma treatment

Hepatocellular carcinoma (HCC) is one of the most fatal solid malignancies worldwide. Evidence suggests that thrombin stimulates tumor progression via fibrin formation and platelet activation. Meanwhile, we also found a correlation between thrombin and HCC through bioinformatics analysis. Dabigatran is a selective, direct thrombin inhibitor that reversibly binds to thrombin. Dabigatran was used as the lead agent in this study, and 19 dabigatran derivatives were designed and synthesized based on docking mode. The thrombin-inhibitory activity of the derivative AX-2 was slightly better than that of dabigatran. BX-2, a prodrug of AX-2, showed a fairly strong inhibitory effect on thrombin-induced platelet aggregation, and effectively antagonized proliferation of HCC tumor cells induced by thrombin at the cellular level. Furthermore, BX-2 reduced tumor volume, weight, lung metastasis, and secondary tumor occurrence in nude mouse models. BX-2 combined with sorafenib increased sorafenib efficacy. This study lays the foundation for discovering new anti-HCC mechanism based on thrombin. BX-2 can be used as an anti-HCC drug lead for further research.

Development of a Rapid Qualitative Screen for Anticoagulant Presence.

Direct oral anticoagulants (DOACs) and fondaparinux with stable pharmacokinetics are commonly used anticoagulants for outpatient care. Due to the lack of monitoring requirements, drug-specific assays are not available in most hospital laboratories, but drug levels are needed in some urgent/emergency situations. This study describes the development of a qualitative screen for the presence of DOAC or fondaparinux using coagulation tests found in most laboratories. The DOAC screen is composed of a heparin anti-Xa activity assay and thrombin time (TT) assay. The STA®-Liquid-Anti-Xa assay calibrated with Stago Multi Hep® and STA®-TT were run on STA-R Max® analyzers. The anti-Xa activity and TT assays were repeated 5 times in samples of commercially available calibrators and controls for each drug: fondaparinux, dabigatran, rivaroxaban, apixaban, and edoxaban. Statistical analysis and correlations were performed for anti-Xa activity and TT results for each drug and pooled normal plasma. A significant correlation was found between heparin-calibrated anti-Xa levels and fondaparinux, rivaroxaban, apixiban, and edoxaban (r2 = 0.99-1.0). Dabigatran showed a strong linear correlation (r2 = 0.99) with TT. Anti-Xa levels >0.3 IU/mL and TT >25 seconds were determined as cutoffs at our lab for the detection of clinically relevant drug levels of factor Xa inhibitor and direct thrombin inhibitor, respectively. Our study demonstrates that commonly available heparin anti-Xa activity and TT assays can be used to qualitatively detect DOACs and fondaparinux and provides a method to establish a qualitative interpretation.

Redesigning apoptotic regulator Bid for thrombin activation

Redesigning apoptotic regulator Bid for thrombin activation

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.

Sustainable inflammatory activation following spinal cord injury is driven by thrombin-mediated dynamic expression of astrocytic chemokines

Acute spinal cord injury (SCI) always results in sustainable recruitment of inflammatory cells driven by sequentially generated chemokines, thereby eliciting excessive neuroinflammation. However, the underlying mechanism of temporally produced chemokines remains elusive. Reactive astrocytes are known to be the main sources of chemokines at the lesion site, which can be immediately activated by thrombin following SCI. In the present study, SCI was shown to induce a sequential production of chemokines CCL2 and CCL5 from astrocytes, which were associated with a persistent infiltration of macrophages/microglia. The rapidly induced CCL2 and later induced CCL5 from astrocytes were regulated by thrombin at the damaged tissues. Investigation of the regulatory mechanism revealed that thrombin facilitated astrocytic CCL2 production through activation of ERK/JNK/NFκB pathway, whereas promoted CCL5 production through PLCβ3/NFκB and ERK/JNK/NFκB signal pathway. Inhibition of thrombin activity significantly decreased production of astrocytic CCL2 and CCL5, and reduced the accumulation of macrophages/microglia at the lesion site. Accordingly, the locomotor function of rats was remarkably improved. The present study has provided a new regulatory mechanism on thrombin-mediated sequential production of astrocytic chemokines, which might be beneficial for clinical therapy of CNS neuroinflammation.

Diversity of Plasmatic Thrombin Pools Regulate Thrombin Generation and Blood Clotting: Interference By a Novel Nanobody

Background: Thrombin has a multitude of roles in the initiation and propagation of (anti)coagulation, in platelet activation and fibrin clot formation. The assessment of thrombin generation (TG) and thrombin-dynamics, combined with multi-omics data, has provided in-depth understanding of the plasma factors contributing to the (in)activation of thrombin in triggered plasma and whole-blood. By default, the generated thrombin is considered to act as a single pool. Here, we present evidence for at least two major proteolytically active thrombin pools with non-overlapping functions in blood clotting. Methods: A novel Syn-Nb-AF106 anti-fibrin nanobody (Nb106) was characterized, targeting a species-conserved thrombin-binding site exposed by fibrinopeptide A cleavage from fibrinogen (Sun et al, this meeting). Thrombin generation was assessed by calibrated automated thrombography using various triggers with control (multi-donor) and (reconstituted) coagulation factor-deficient plasmas, platelet-rich plasma or whole blood. Fibrin clot formation was evaluated by scanning electron microscopy (SEM). Clot retraction was macroscopically assessed. Results: In tissue factor-triggered thrombin generation,we discovered that Nb106 dose-dependently reduced the thrombin peak level and endogenous thrombin potential (ETP) to 54-62%, without affecting kinetic thrombin generation parameters. We observed a similar reduction in platelet-free plasma, platelet-rich plasma and whole blood. Biochemically, Nb106 displaced thrombin from a binding site on cleaved fibrinogen (Sun et al., abstract). Nb106 inhibited tissue factor-induced thrombin generation, using plasma deficient in factor IX, XI or XII, but not using plasmas deficient in fibrinogen or antithrombin. Reconstitution experiments showed that the inhibition depended on the fibrinogen concentration. A-specific control nanobodies were without effect. Together, the fibrin(ogen)-dependent and thrombin-inhibiting effect indicated that Nb106 operates by releasing proteolytically-active thrombin from fibrin to allow inactivation by antithrombin. Time-dependent, late spiking experiments revealed that Nb106 reduced the generation of thrombin only when added at 0-15 minutes after trigger, but no longer after 30 minutes, suggesting that the inhibitory capacity was due to a shortened thrombin binding to fibrin rather than to a continued protection towards antithrombin. In plasmas from a cohort of 64 healthy subjects, application of Nb106 showed a consistent reducing effect on the parameters peak and ETP, with a moderate positive correlation with the fibrinogen level. Nb106 suppressed the thrombin generation amplitude, but not the kinetics in the presence of a panel of direct oral anticoagulants (DOAC). Strikingly, brightfield microscopy and SEM indicated that Nb106, but not control nanobodies, in triggered plasma, platelet-rich plasma and whole-blood, fully abrogated the formation of elongating fibrin fibers. In agreement with this, it strongly suppressed whole-blood clot retraction. Conclusion: Collectivity, these data point to the existence of two major pools of proteolytically active thrombin, both of which contributing to thrombin generation, that are formed upon plasma and blood coagulation. One pool of soluble thrombin steers the proteolytic coagulation cascade, while another pool is bound to growing fibrin fibers. The latter pool still cleaves the conventional AMC thrombin substrate, is temporarily protected against antithrombin, and is required for fibrin fiber extension and blood clotting and clot retraction. The Nb106, replacing thrombin from its binding site on fibrin, selectively annuls the second pool, thereby blocking the clotting process. The replacement ensures thrombin inactivation by antithrombin. Perspective: The novel anti-fibrin nanobody Syn-Nb-AF106 or a derivative has the potential to act as an alternative anticoagulant in thrombotic diseases with pathological fibrin formation.

A Mouse Model of the PAR4 Sequence Variant P310L Shows Decreased Platelet Reactivity and Thrombosis

Protease activated receptors 1 and 4 (PAR1, PAR4) on platelets mediate thrombin signaling and are vital for thrombus development. Hypercoagulation and thrombin generation are major risk factors for thrombosis. Sustained thrombin signaling through PAR4 promotes platelet activation, phosphatidylserine exposure, and subsequent thrombin generation. A single-nucleotide polymorphism (SNP, rs2227376) leads to an amino acid change in extracellular loop 3 (ECL3) of PAR4, which decreases PAR4 activation (PAR4-P310L). This SNP is also associated with a significantly lower risk for venous thromboembolism (VTE) in a GWAS meta-analysis. We aim to determine the mechanism by which PAR4-P310L (rs2227376) impacts platelet function and reduces the risk of VTE using mice with a homologous mutation (PAR4-P322L). Ex vivo platelet function was determined using flow cytometry and aggregometry. Arterial and venous thrombosis was examined using the ferric-chloride induced carotid artery injury and inferior vena cava (IVC) stasis models, respectively. Platelets from heterozygous (PAR4-322P/L) and homozygous (PAR4-322L/L) mice had decreased thrombin-mediated aggregation and decreased PAR4 reactivity compared to wild types. These mice also had a prolonged time to occlusion in arterial thrombosis, with 55% of PAR4-322P/L mice unable to develop stable thrombi within 30 minutes. In venous thrombosis, wild-type mice developed clotted IVCs that averaged 31 mg after 48 hours, while global PAR4-knockout mice averaged 21 mg (p<0.01), PAR4-322P/L 23 mg (p<0.01), and PAR4-322L/L 28 mg. Since platelet procoagulant activity is known to drive venous thrombosis, we used lactadherin to show that PAR4-P322L platelets express less phosphatidylserine in response to thrombin (5 nM) and convulxin (1 nM). Platelet-rich plasma from PAR4-P322L mice also generated less thrombin compared to wild-types, and whole blood from mice lacking PAR4 showed delayed clot formation in rotational thromboelastometry. Altogether, this suggests a direct impact of PAR4 signaling on platelet-mediated thrombin generation. Our new mouse model shows that the polymorphism in ECL3, PAR4-322L, decreases platelet reactivity and thrombosis. We also show PAR4-322L leads to less procoagulant platelets and decreases the endogenous thrombin potential. This decreased ability to generate thrombin offers a mechanism for PAR4's role in VTE. Our findings highlight a key role of PAR4 signaling in platelet activation and procoagulant activity in thrombosis.

Fibrinolysis Assessment with Tpa-ROTEM in Patients with Bleeding Disorders of Unknown Cause (BDUC)

Background: In 40-70% of patients referred to the hematologist for evaluation of bleeding symptoms, no cause for their increased bleeding tendency can be found using currently available hemostasis laboratory tests. This group is classified as patients with bleeding disorder of unknown cause (BDUC). The lack of clear guidelines concerning diagnosis, management and genetic counseling in BDUC-patients poses a challenge for patients, family members and treating physicians. Several hemostasis assays have been investigated in BDUC-patients, but little is known about the role of the fibrinolytic pathway in this patient group. This can be explained by the lack of standardized fibrinolysis tests and the limited availability of these tests in laboratories. To further unravel potential bleeding mechanisms in BDUC, in the present study we assessed fibrinolysis in BDUC patients by using a global whole blood tPA ROTEM assay and determining of individual fibrinolysis proteins. Aim: To investigate the capacity of the tPA-ROTEM assay to detect fibrinolytic abnormalities in BDUC-patients and to correlate these findings with fibrinolytic protein activity levels in plasma. Methods: Data from patients referred to the hematologist for bleeding evaluation and included in the Prediction of Evaluation in Adult Hematologic Patients with Bleeding Tendencies (ProBe-AHP) study (METC:14-4-036) were used. BDUC-patients were identified based on (1) a clinically relevant increased bleeding tendency and (2) no abnormalities in extended hemostasis laboratory testing. tPA-ROTEM assay was done according to the in 2016 validated and published method on the ROTEM device. Fibrinolysis protein plasma levels, including plasminogen, plasminogen activator inhibitor 1 (PAI-1) antigen and activity, tissue plasminogen activator (tPA) antigen, alpha2-antiplasmin and thrombin activatable fibrinolysis inhibitor (TAFI), were measured using ELISA assays. Pre-specified definitions of hyper- and hypofibrinolysis as measured by tPA-ROTEM were applied to identify hyper- and hypofibrinolytic profiles. Results: tPA-ROTEM identified a hyperfibrinolytic profile in 15/73 (20%) of BDUC-patients, a hypofibrinolytic profile in 16/73 (22%) of BDUC-patients and a normal profile in 42 BDUC-patients (58%). Plasminogen activity inhibitor 1 (PAI-1) activity and antigen levels and α2-antiplasmin levels were lower in BDUC-patients with a hyperfibrinolytic profile compared to BDUC-patients with a hypofibrinolytic profile. No statistically significant correlations between tPA-ROTEM parameters and fibrinolysis protein plasma levels were identified using multivariate regression analysis. Conclusion: tPA-ROTEM identified a hyperfibrinolytic profile in 20% of BDUC-patients. In this group, the lower PAI-1 activity and antigen levels and lower α2-antiplasmin levels are in concordance with the expected changes in case of hyperfibrinolysis. These findings indicate that these fibrinolysis protein plasma levels are within the lower functional range of their distribution, shifting the balance towards increased clot lysis leading to an increased bleeding tendency. These findings might deliver evidence to the use of antifibrinolytic therapies in BDUC-patients in case of bleeding or interventions. Informed consent and approval from the medical-ethical committee from Maastricht (METC azM/UM) were obtained.

Deciphering Thrombosis Risk in Vexas Syndrome Patients through Thrombin Generation Assay Andthromboelastography

Introduction: VEXAS syndrome (for Vacuoles in myeloid progenitors, E1 ubiquitin ligase, X-linked, Autoinflammatory manifestations and Somatic) is the consequence of the clonal expansion of hematopoietic stem or progenitor cells with somatically acquired mutation of UBA1. VEXAS is a rare disease characterized by a variety of autoinflammatory manifestations and frequently associated with other hematological malignancies. Venous (VTE) and arterial thrombosis (AT) are recurrently observed in disease course and may affect up to 50% of patients. The exact pathophysiological mechanism underlying thrombosis genesis in UBA1 mut patients is not yet elucidated. Thrombin generation assay (TGA) and thromboelastography (ROTEM) are global hemostasis assays that might be used to identify hemostatic profiles at risk of thrombosis (Ay C et al. J Clin Oncol 2011). In this study, we aimed to evaluate TGA and ROTEM as potential surrogate markers of clot formation in UBA1 mut patients to predict their risk for thrombosis. Methods: TGA and ROTEM assays were performed in UBA1 mut patients between March 2022 and July 2023 , in Hospices Civils de Lyon, France. Patients were tested whether or not they had a personal history of VTE/AT. For patients already treated with oral anticoagulant interfering with exploratory assays, a temporary heparin switch was realized. TGA allows to evaluate concentration of generated thrombin through time, resulting in a dynamic thrombin generation curve, from which various parameters can be determined that describe thrombin activity, such as endogenous thrombin potential (ETP; area under the TG curve). Regarding ROTEM, analyses have been performed on citrated tubes using the ROTEM delta. For each test, the parameters CT (clotting time in seconds), A5 and A10 (measured clot amplitude at 5 and 10 minutes in mm), angle alpha (velocity in °), maximum clot firmness (in mm) and LI30 (% clot lysis at 30 minutes) have been measured. Results: We screened 15 UBA1 mut patients, while only 14 were evaluable for TGA/ROTEM (1 patient excluded due to concomitant use of apixaban the day of TGA). Five out of 15 (33%) had an history of VTE (N=3) or AT (N=2) at the time of TGA/ROTEM blood tests. With a median follow-up of 11.8 months after hmostastic explorations, none of screened patients experienced VTE/AT. On the 14 evaluable patients, TGA/ROTEM exploration was performed in 4 of them at the time of diagnosis prior any treatment onset (including steroids). Active disease (defined by increased CRP and/or clinical manifestations) at the time of TGA did not influence significantly time to peak (mins), peak thrombin (nM) and endogeneous thrombin potential (ETP, nM. min) either in platelet rich- and platelet poor- plasma. Interestingly, patients with prior history of VTE/AT (N=4) had significantly higher ETP compared to those without thrombosis history (N=10) either in platelet rich- and platelet poor-plasmas. Moreover, UBA1 mut patients without thrombosis history exhibited similar ETP values than controls ( figure 1). There was no significant correlation between type of UBA1 mutation, UBA1 allelic frequency, concomitant MDS, type of treatment at the time of TGA assay. Regarding ROTEM analysis, there was no significant differences in clot formation between patients with or without thrombosis history in EXTEM, NATEM, and FIBTEM channels. However, in TEMACT channel (activation as in EXTEM but with a plasminogen activator), we observed higher MCF (Maximum Clot Firmness) in UBA1 mutpatients with prior history of VTE/AT ( Figure 2). Acquired and inherited thrombophilia (protein C and S activities, lupus anticoagulant, anticardiolipine antibodies, anti-beta2 GP1 antibodies, factor V Leiden and prothrombin G20210A mutations, antithrombin evels) were explored in 8 out 15 of our patients. Two of them harbored lupus anticoagulant but without antiphospholipid antibodies. They do not have eitherthrombosis history or higher ETP levels. Conclusions: Based on these preliminary results, TGA and more specifically ETP might identify UBA1 mutpatients at risk of VTE or AT. ROTEM identified an hypofibrinolytic phenotype UBA1 mutpatients with thrombosis history paving the way for future explorations. The small sample size and the retrospective (post-VTE/AT) nature of these explorations are main limitations of this study, which need to be confirmed with a larger follow-up.

Evolution Directed Gene Engineering (EDGE) Identifies Single Amino Acid Substitutions in FVIII with Six-Fold Increased in Vitro and In Vivo Activity

Background: Current AAV gene therapy for hemophilia A (HA) is limited by low sustained factor VIII (FVIII) levels. In contrast, AAV gene therapy for hemophilia B benefits from the widespread adoption of the single amino acid (AA) substituted high-specific-activity FIX variant R338L. We have previously shown that wild-type (WT) FIX has not evolved for maximal specific activity, but rather has evolutionary conserved AAs that limit procoagulant activity (Samelson-Jones et al. Blood Advances 2021). FVIII may have evolved with similar considerations. Based on this work, we developed a novel screening technique, termed Evolution Directed Gene Engineering (EDGE), to identify new hyperactive single AA substituted FVIII variant transgenes to address the current limitations of HA gene therapy. Aims: To find and characterize single AA substituted FVIII variants that can enhance gene transfer. Methods: EDGE evaluates AA positions fulfilling 2 criteria: 1) highly evolutionary conserved and 2) not associated with the disease phenotype. Here, EDGE compared 33 mammalian FVIII orthologues and then eliminated positions associated with known HA-causing missense mutations. FVIII variants were transiently expressed in Huh7 cells and cultured media was assayed for FVIII activity and antigen. Recombinant FVIII protein of lead candidates were purified to homogeneity and assayed for FVIII activity with one- and two-stage aPTT assays, thrombin generation assay (TGA), and enzyme kinetics studies. The in vivo efficacy of the variants was tested by tail clip assays of HA mice after retro-orbital delivery of the protein or liver-directed AAV gene therapy. Results: Out of 52 AA positions identified by EDGE, we found 12 AA positions in FVIII where single AA substitutions increased specific activity ≥2-fold (Figure 1A). The largest increase in specific activity was 6-fold, which occurred for multiple AA substitutions at position 659. Purified recombinant protein of FVIII-K659M and -K659V both demonstrated 6-fold increased activity in one- and two-stage aPTT assays, but comparable thrombin activation and A2-stability to FVIII-WT. TGA show the FVIII-K659M/V variants have greater than 6-fold improvements in endogenous thrombin potential and peak thrombin generation. Compared to FVIII-WT, FVIII-K659M/V proteins have an increased k cat and decreased appK D which suggests that the mechanism of increased specific activity is due to enhanced interactions with FIXa. This is similar to the mechanism of hyperactivity of FIX-R338L. FVIII-K659M/V also demonstrated enhanced procoagulant activity in vivo. In tail clip injuries of HA mice (n≥4) treated with purified protein, the FVIII-K659M/V proteins restored normal hemostasis at 5-fold lower concentrations than FVIII-WT. When mice were treated with equal doses of 2.5 µg/kg of protein, FVIII-K659M/V restored normal hemostasis while FVIII-WT had no significant change in blood loss (Figure 1B). As expected, 12.5 µg/kg of FVIII-WT protein was required to restore blood loss to WT mouse levels. FVIII-K659 substituted variants also enhanced AAV based gene therapy. HA mice (n=6) treated with AAV8 vectors expressing FVIII-K659C had 6-fold higher plasma FVIII activity than mice (n=6) treated at the same dose with AAV8 vectors expressing FVIII-WT. HA mice that received 4 x 10 12 vg/kg of AAV8 FVIII-K659C had less bleeding than HA mice that received the same dose of AAV8 FVIII-WT, but similar blood loss as WT mice after tail clip injury. Conclusions: The success of EDGE in identifying multiple new high specific activity FVIII variants supports the hypothesis that FVIII-WT is not optimized for specific activity and thus is amenable to bioengineering approaches. The ≥5-fold enhanced in vitro and in vivo hemostatic activity of the FVIII-K659 substituted variants and initial murine gene therapy studies support their translational potential to address current limitations of HA gene therapy. These results also implicate K659 in modulating the FVIIIa/FIXa interaction. EDGE may also be applicable to other gene therapy targets as well.

Endothelial dysfunction as predictor of atherosclerosis during a translatitudinal sea voyage in the Arctic: prospective study

INTRODUCTION. Human labor activity in the Arctic takes place in severe climatic conditions associated with cooling, atmospheric pressure drops, high humidity, photoperiodicity, low oxygen content in the air. Endothelial dysfunction is an early pathophysiological sign and an independent predictor of poor prognosis in most cardiovascular diseases.
 OBJECTIVE.Тo analyze the dynamics of the level of metalloproteinase-9 and thrombin activity as predictors of the development of atherosclerosis in sailors during a translatitudinal sea voyage in the Arctic.
 MATERIALS AND METHODS. A prospective clinical and laboratory study of crew members was performed during the integrated marine research expedition “Transarctic-2019”. A clinical and laboratory study of the level of metalloprotease-9 (enzyme-linked immunosorbent assay), coagulation analysis of the parameters of the thrombin generation test, as well as ultrasonic dopplerography of the thickness of the intima-media complex were carried out. Statistical data processing was carried out using the methods of descriptive and analytical statistics in the StataCorp Stata 14.2 program. The study protocol was approved by the local ethical committee of the SSMU (protocol No. 03/5 dated May 27, 2015) and supported by the state task reg. No. NIOKTR 121030300111-7.
 RESULTS. A statistically significant increase in the level of MMP-9 in sailors after the expedition was noted, the relationship between the level of MMP-9, indicators of the thrombin generation test (lag time, min (p=0.0190), tPeak (p=0.0177), Peak (p= 0.0217) and KIM thickening.
 DISCUSSION. MMP-9 levels may be a predictor of early atherosclerosis and atherosclerotic plaque instability, as well as a risk factor for future adverse cardiovascular events. The relationship of MMP-9 with the kinetics of thrombin (one of the regulators of the antithrombotic state of the endothelium) during a translatitudinal voyage in sailors indicates the formation of readiness for prothrombotic events.
 CONCLUSION. Thus, in Arctic conditions, under conditions of chronic oxidative stress, which contribute to the progression of the development of endothelial dysfunction in the presence of external risk factors, the likelihood of progression of atherosclerotic vascular lesions increases, and, as a result, the development of vascular events.

Effects of the activated coagulation factor X (FXa) and its protease-activated receptor-2 (PAR2) on colon cancer cell growth in vitro and in vivo

Abstract Background Protease-activated receptors are a family of four (PAR1-4) G-protein-coupled receptors with a unique mechanism of activation. Serine proteases such as the activated coagulation factor X (FXa) can activate PARs, selectively PAR2, by proteolytic cleavage unmasking its tethered ligand which induces consecutive receptor activation. PAR2 has been linked to various pathophysiological processes such as cardiovascular or inflammatory diseases. Recent studies indicate a key role of PAR2 in certain cancer types. Purpose In this study, we investigated the impact of FXa, PAR2 and of the direct, oral FXa-inhibitor Apixaban in colon cancer cell growth and migration in vitro and in vivo. Methods FXa and PAR2-dependent effects were investigated in various colon cancer cell lines such as human HCT116 or murine MC38 cells in vitro using different assays for cell proliferation and migration, Western blotting and PCR analyses. A subcutaneous tumor model was utilized to compare the impact of Apixaban (5 and 50 mg/kg body weight) in C57Bl/6 wild type (WT) versus PAR2-deficient mice (PAR2-KO). Tumor cells were allowed to grow for up to 21 days. Apixaban was applied orally by gavage. All animal studies were approved by the responsible animal welfare committee. Results Incubation with FXa at concentrations up to 30 nM stimulated growth and migration in both cultured human and murine cells in vitro. These effects were mimicked by peptide-stimulated activation of PAR2. In comparison, thrombin or selective peptide-induced activation of PAR1 did not induce a marked response. In addition, we observed an activation of mitogenic signaling pathways such as p44/42, p38 and AKT by FXa suggesting a direct response for colon cancer cells to coagulation factor signaling. In vivo, PAR2-deficiency resulted in a significantly reduced tumor growth compared to wild-type mice. In comparison, oral treatment with Apixaban both at 5 mg or 50 mg/kg body weight did not markedly affect tumor development in vivo. This effect was reflected by a reduced tumor weight in PAR2-deficient mice after excision of the tumor at the end of the study. In addition, a significantly lower rate of complications was seen in PAR2-deficient mice during the experiment. Interestingly, the spleen was larger in relation to body weight in PAR2-deficient mice independently from Apixaban treatment. We observed no obvious histological differences within the tumor tissue between the treatment groups or animal strains. Conclusion Deficiently of PAR2 resulted in reduced growth of colon cancer cells in vivo, while oral treatment with Apixaban did not significantly affect tumor growth. In vitro, FXa and selective activation of PAR2 did stimulate cell growth and migration indicating a potential direct growth promotion effect of PAR2 in colon cancer cells. The pharmacological targeting of PAR2 may represent a future therapeutic strategy in the treatment of colon cancer.