Glycated albumin modulates the contact system with implications for the kallikrein-kinin and intrinsic coagulation systems.

BACKGROUND: Human serum albumin (HSA) is the most abundant plasma protein and is sensitive to glycation in vivo. The chronic hyperglycemic conditions in patients with diabetes mellitus (DM) induce a nonenzymatic Maillard reaction that denatures plasma proteins and forms advanced glycation end products (AGEs). HSA-AGE is a prevalent misfolded protein in patients with DM and is associated with factor XII activation and downstream proinflammatory kallikrein-kinin system activity without any associated procoagulant activity of the intrinsic pathway. OBJECTIVES: This study aimed to determine the relevance of HSA-AGE toward diabetic pathophysiology. METHODS: The plasma obtained from patients with DM and euglycemic volunteers was probed for activation of FXII, prekallikrein (PK), and cleaved high-molecular-weight kininogen by immunoblotting. Constitutive plasma kallikrein activity was determined via chromogenic assay. Activation and kinetic modulation of FXII, PK, FXI, FIX, and FX via in vitro-generated HSA-AGE were explored using chromogenic assays, plasma-clotting assays, and an in vitro flow model using whole blood. RESULTS: Plasma obtained from patients with DM contained increased plasma AGEs, activated FXIIa, and resultant cleaved cleaved high-molecular-weight kininogen. Elevated constitutive plasma kallikrein enzymatic activity was identified, which positively correlated with glycated hemoglobin levels, representing the first evidence of this phenomenon. HSA-AGE, generated in vitro, triggered FXIIa-dependent PK activation but limited the intrinsic coagulation pathway activation by inhibiting FXIa and FIXa-dependent FX activation in plasma. CONCLUSION: These data indicate a proinflammatory role of HSA-AGEs in the pathophysiology of DM via FXII and kallikrein-kinin system activation. A procoagulant effect of FXII activation was lost through the inhibition of FXIa and FIXa-dependent FX activation by HSA-AGEs.

Novel interaction of properdin and coagulation factor XI: Crosstalk between complement and coagulation.

Background: Evidence of crosstalk between the complement and coagulation cascades exists, and dysregulation of either pathway can lead to serious thromboinflammatory events. Both the intrinsic pathway of coagulation and the alternative pathway of complement interact with anionic surfaces, such as glycosaminoglycans. Hitherto, there is no evidence for a direct interaction of properdin (factor P [FP]), the only known positive regulator of complement, with coagulation factor XI (FXI) or activated FXI (FXIa). Objectives: The aim was to investigate crosstalk between FP and the intrinsic pathway and the potential downstream consequences. Methods: Chromogenic assays were established to characterize autoactivation of FXI in the presence of dextran sulfate (DXS), enzyme kinetics of FXIa, and the downstream effects of FP on intrinsic pathway activity. Substrate specificity changes were investigated using SDS-PAGE and liquid chromatography-mass spectrometry (LC-MS). Surface plasmon resonance (SPR) was used to determine direct binding between FP and FXIa. Results/Conclusions: We identified a novel interaction of FP with FXIa resulting in functional consequences. FP reduces activity of autoactivated FXIa toward S-2288. FXIa can cleave FP in the presence of DXS, demonstrated using SDS-PAGE, and confirmed by LC-MS. FXIa can cleave factor IX (FIX) and FP in the presence of DXS, determined by SDS-PAGE. DXS alone modulates FXIa activity, and this effect is further modulated by FP. We demonstrate that FXI and FXIa bind to FP with high affinity. Furthermore, FX activation downstream of FXIa cleavage of FIX is modulated by FP. These findings suggest a novel intercommunication between complement and coagulation pathways.

Drugs in phase I and II clinical development for the prevention of stroke in patients with atrial fibrillation.

INTRODUCTION: Atrial fibrillation is the most frequently diagnosed cardiac arrhythmia globally and is associated with ischemic stroke and heart failure. Patients with atrial fibrillation are typically prescribed long-term anticoagulants in the form of either vitamin K antagonists or non-vitamin K antagonist oral anticoagulants; however, both carry a potential risk of adverse bleeding. AREAS COVERED: This paper sheds light on emerging anticoagulant agents which target clotting factors XI and XII, or their activated forms - XIa and XIIa, respectively, within the intrinsic coagulation pathway. The authors examined data available on PubMed, Scopus, and the clinical trials registry of the United States National Library of Medicine (www.clinicaltrials.gov). EXPERT OPINION: Therapies targeting factors XI or XII can yield anticoagulant efficacy with the potential to reduce adverse bleeding. Advantages for targeting factor XI or XII include a wider therapeutic window and reduced bleeding. Long-term follow-up studies and a greater understanding of the safety and efficacy are required. Atrial fibrillation is a chronic disease and therefore the development of oral formulations is key.

Kallikrein directly interacts with and activates Factor IX, resulting in thrombin generation and fibrin formation independent of Factor XI.

Kallikrein (PKa), generated by activation of its precursor prekallikrein (PK), plays a role in the contact activation phase of coagulation and functions in the kallikrein-kinin system to generate bradykinin. The general dogma has been that the contribution of PKa to the coagulation cascade is dependent on its action on FXII. Recently this dogma has been challenged by studies in human plasma showing thrombin generation due to PKa activity on FIX and also by murine studies showing formation of FIXa-antithrombin complexes in FXI deficient mice. In this study, we demonstrate high-affinity binding interactions between PK(a) and FIX(a) using surface plasmon resonance and show that these interactions are likely to occur under physiological conditions. Furthermore, we directly demonstrate dose- and time-dependent cleavage of FIX by PKa in a purified system by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and chromogenic assays. By using normal pooled plasma and a range of coagulation factor-deficient plasmas, we show that this action of PKa on FIX not only results in thrombin generation, but also promotes fibrin formation in the absence of FXII or FXI. Comparison of the kinetics of either FXIa- or PKa-induced activation of FIX suggest that PKa could be a significant physiological activator of FIX. Our data indicate that the coagulation cascade needs to be redefined to indicate that PKa can directly activate FIX. The circumstances that drive PKa substrate specificity remain to be determined.

Observations on clot properties in atrial fibrillation: Relation to renal function and choice of anticoagulant.

INTRODUCTION: Atrial fibrillation (AF) is associated with increased risk of stroke and thromboembolism. Patients with AF have a higher incidence of renal impairment, which may influence the risks of systemic thromboembolism or bleeding. We determined how different oral anticoagulants affect plasma clot properties and whether progressive renal dysfunction affects plasma clot properties in patients on warfarin. MATERIALS AND METHODS: We studied 257 patients with AF receiving oral anticoagulants. Furthermore, we recruited 192 separate patients with AF on warfarin and divided them in 4 groups based on estimated glomerular filtration rate (eGFR). Platelet poor plasma was prepared and clot formation and fibrinolysis was monitored kinetically up to 1 h. RESULTS: Rate of clot formation was significantly slower with dabigatran and rivaroxaban. Time between 50% clotting and 50% lysis was prolonged in patients receiving warfarin compared to NOACs. Time to 50% lysis from maximum absorbance was significantly shorter in patients receiving rivaroxaban. Time between 50% clotting and 50% lysis became significantly prolonged with worsening eGFR. Time to 50% lysis from maximum absorbance was prolonged as renal function worsened. CONCLUSIONS: Compared to warfarin, NOACs differently modulate coagulation and fibrinolysis under ex vivo conditions. Worsening renal function in AF patients on warfarin prolongs fibrinolysis, potentially increasing the risk of thrombosis.

Evaluation of the Total Thrombus-Formation System (T-TAS): application to human and mouse blood analysis.

The Total Thrombus-formation Analyser System (T-TAS) is a whole blood flow chamber system for the measurement of in vitro thrombus formation under variable shear stress conditions. Our current study sought to evaluate the potential utility of the T-TAS for the measurement of thrombus formation within human and mouse whole blood. T-TAS microchips (collagen, PL chip; collagen/tissue thromboplastin, AR chip) were used to analyze platelet (PL) or fibrin-rich thrombus formation, respectively. Blood samples from humans (healthy and patients with mild bleeding disorders) and wild-type (WT), mice were tested. Light transmission lumi-aggregometer (lumi-LTA) was performed in PRP using several concentrations of ADP, adrenaline, arachidonic acid, collagen, PAR-1 peptide and ristocetin. Thrombus growth (N = 22) increased with shear within PL (4:40 ± 1.11, 3:25 ± 0.43 and 3:12 ± 0.48 mins [1000, 1500 and 2000s-1]) and AR chips (3:55 ± 0.42 and 1:49 ± 0.19 [240s-1 and 600s-1]). The area under the curve (AUC) on the PL chip was also reduced at 1000s-1 compared to 1500/2000s-1 (260 ± 51.7, 317 ± 55.4 and 301 ± 66.2, respectively). In contrast, no differences in the AUC between 240s-1 and 600s-1 were observed in the AR chip (1593 ± 122 and 1591 ± 158). The intra-assay coefficient of variation (CV) (n = 10) in the PL chip (1000s-1) and AR chip (240s-1) were T1014.1%, T6016.7%, T10-6022.8% and AUC1024.4% or T10 9.03%, T808.64%, T10-8023.8% and AUC305.1%. AR chip thrombus formation was inhibited by rivaroxaban (1 µM), but not with ticagrelor (10 µM). In contrast, PL chip thrombus formation was totally inhibited by ticagrelor. T-TAS shows an overall agreement with lumi-LTA in 87% of patients (n = 30) with normal PL counts recruited into the genotyping and phenotyping of platelet (GAPP) study and suspected to have a PL function defect. The onset (T10) of thrombus formation in WT mice (N = 4) was shorter when compared to humans e.g. PL chip (1000s-1) T10 were 02:02 ± 00:23 and 03:30 ± 0:45, respectively). T-TAS measures in vitro thrombus formation and can be used for monitoring antithrombotic therapy, investigating patients with suspected PL function defects and monitoring PL function within mice.

The role of activated coagulation factor XII in overall clot stability and fibrinolysis.

Activated coagulation factor XII (α-FXIIa) is able to bind to fibrin(ogen) and increases the density and stiffness of the fibrin clot. Conversely, proteins of the contact system and the fibrinolytic system show a high degree of homology and α-FXIIa can convert plasminogen into plasmin resulting in fibrin degradation. Therefore, we studied the contribution of α-FXIIa to overall clot stability and plasmin driven fibrinolysis in the absence and presence of tissue plasminogen activator (tPA). We observed that α-FXIIa directly converted plasminogen into plasmin and reduced clot lysis time at all tPA concentrations tested (15-1500 pM). Simultaneous assessment of plasmin generation (chromogenic substrate S-2251) and fibrin formation and degradation (absorbance at 405nm), showed an earlier onset of fibrinolysis and plasmin formation in the presence of α-FXIIa. Fibrinolysis of clots formed under flow conditions, revealed that incorporation of α-FXIIa accelerated clot breakdown (fluorescence release of labeled fibrin) by additional plasmin generation on top of formation by tPA. Scanning electron microscopy (SEM) revealed that the surface area pore size increased in the presence compared with the absence of α-FXIIa when fibrinolysis was initiated by the conversion of plasminogen with tPA during clot formation. α-FXIIa enhances fibrinolysis in the presence of plasminogen, irrespective of whether tPA was present during clot formation or was added afterwards to initiate fibrinolysis. We postulate that FXIIa first strengthens the clot structure during clot formation and thereafter contributes towards fibrinolysis.