Pretreatment with a dual antiplatelet and anticoagulant (APAC) reduces ischemia-reperfusion injury in a mouse model of temporary middle cerebral artery occlusion-implications for neurovascular procedures.

BACKGROUND: Several neurovascular procedures require temporary occlusion of cerebral arteries, leading to ischemia of unpredictable length, occasionally causing brain infarction. Experimental models of cerebral ischemia-reperfusion injury have established that platelet adhesion and coagulation play detrimental roles in reperfusion injury following transient cerebral ischemia. Therefore, in a model of cerebral ischemia-reperfusion injury (IRI), we investigated the therapeutic potential of a dual antiplatelet and anticoagulant (APAC) heparin proteoglycan mimetic which is able to bind to vascular injury sites. METHODS: Brain ischemia was induced in mice by transient occlusion of the right middle cerebral artery for 60 min. APAC, unfractionated heparin (UFH) (both at heparin equivalent doses of 0.5 mg/kg), or vehicle was intravenously administered 10 min before or 60 min after the start of ischemia. At 24 h later, mice were scored for their neurological and motor behavior, and brain damage was quantified. RESULTS: Both APAC and UFH administered before the onset of ischemia reduced brain injury. APAC and UFH pretreated mice had better neurological and motor functions (p < 0.05 and p < 0.01, respectively) and had significantly reduced cerebral infarct sizes (p < 0.01 and p < 0.001, respectively) at 24 h after transient occlusion compared with vehicle-treated mice. Importantly, no macroscopic bleeding complications were observed in either APAC- or UFH-treated animals. However, when APAC or UFH was administered 60 min after the start of ischemia, the therapeutic effect was lost, but without hemorrhaging either. CONCLUSIONS: Pretreatment with APAC or UFH was safe and effective in reducing brain injury in a model of cerebral ischemia induced by transient middle cerebral artery occlusion. Further studies on the use of APAC to limit ischemic injury during temporary occlusion in neurovascular procedures are indicated.

Intravenously administered APAC, a dual AntiPlatelet AntiCoagulant, targets arterial injury site to inhibit platelet thrombus formation and tissue factor activity in mice.

INTRODUCTION: Arterial thrombosis is the main underlying mechanism of acute atherothrombosis. Combined antiplatelet and anticoagulant regimens prevent thrombosis but increase bleeding rates. Mast cell-derived heparin proteoglycans have local antithrombotic properties, and their semisynthetic dual AntiPlatelet and AntiCoagulant (APAC) mimetic may provide a new efficacious and safe tool for arterial thrombosis. We investigated the in vivo impact of intravenous APAC (0.3-0.5 mg/kg; doses chosen according to pharmacokinetic studies) in two mouse models of arterial thrombosis and the in vitro actions in mouse platelets and plasma. MATERIALS AND METHODS: Platelet function and coagulation were studied with light transmission aggregometry and clotting times. Carotid arterial thrombosis was induced either by photochemical injury or surgically exposing vascular collagen after infusion of APAC, UFH or vehicle. Time to occlusion, targeting of APAC to the vascular injury site and platelet deposition on these sites were assessed by intra-vital imaging. Tissue factor activity (TF) of the carotid artery and in plasma was captured. RESULTS: APAC inhibited platelet responsiveness to agonist stimulation (collagen and ADP) and prolonged APTT and thrombin time. After photochemical carotid injury, APAC-treatment prolonged times to occlusion in comparison with UFH or vehicle, and decreased TF both in carotid lysates and plasma. Upon binding from circulation to vascular collagen-exposing injury sites, APAC reduced the in situ platelet deposition. CONCLUSIONS: Intravenous APAC targets arterial injury sites to exert local dual antiplatelet and anticoagulant actions and attenuates thrombosis upon carotid injuries in mice. Systemic APAC provides local efficacy, highlighting APAC as a novel antithrombotic to reduce cardiovascular complications.

Urinary extracellular vesicles carry multiple activators and regulators of coagulation.

Cells shape their extracellular milieu by secreting intracellular products into the environment including extracellular vesicles which are lipid-bilayer limited membrane particles. These vesicles carry out a range of functions, including regulation of coagulation, via multiple contributor mechanisms. Urinary extracellular vesicles are secreted by various cells, lining the urinary space, including the nephron and bladder. They are known to have procoagulant properties, however, the details of this function, beyond tissue factor are not well known. The aim of the study was to access the role of urinary extracellular vesicles in impacting coagulation upon supplementation to plasma. This could indicate their physiological function upon kidney injury or pathology. Supplementation to standard human plasma and plasmas deficient in various coagulation factors was used for this purpose, and calibrated automated thrombogram (CAT®) was the major technique applied. We found that these vesicles contain multiple coagulation-related factors, and their lipid composition affects coagulation activities of plasma upon supplementation. Remarkably, these vesicles can restore thrombin generation in FVII, FVIII, FIX and FXI -deficient plasmas. This study explores the multiple roles of urinary extracellular vesicles in coagulation in in vitro blood coagulation and implies their importance in its regulation by several mechanisms.

Thrombosis and Inflammation-A Dynamic Interplay and the Role of Glycosaminoglycans and Activated Protein C.

Hemostasis, thrombosis, and inflammation are tightly interconnected processes which may give rise to thrombo-inflammation, involved in infectious and non-infectious acute and chronic diseases, including cardiovascular diseases (CVD). Traditionally, due to its hemostatic role, blood coagulation is isolated from the inflammation, and its critical contribution in the progressing CVD is underrated, until the full occlusion of a critical vessel occurs. Underlying vascular injury exposes extracellular matrix to deposit platelets and inflammatory cells. Platelets being key effector cells, bridge all the three key processes (hemostasis, thrombosis, and inflammation) associated with thrombo-inflammation. Under physiological conditions, platelets remain in an inert state despite the proximity to the endothelium and other cells which are decorated with glycosaminoglycan (GAG)-rich glycocalyx (GAGs). A pathological insult to the endothelium results in an imbalanced blood coagulation system hallmarked by increased thrombin generation due to losses of anticoagulant and cytoprotective mechanisms, i.e., the endothelial GAGs enhancing antithrombin, tissue factor pathway-inhibitor (TFPI) and thrombomodulin-protein C system. Moreover, the loss of GAGs promotes the release of mediators, such as von Willebrand factor (VWF), platelet factor 4 (PF4), and P-selectin, both locally on vascular surfaces and to circulation, further enhancing the adhesion of platelets to the affected sites. Platelet-neutrophil interaction and formation of neutrophil extracellular traps foster thrombo-inflammatory mechanisms exacerbating the cardiovascular disease course. Therefore, therapies which not only target the clotting mechanisms but simultaneously or independently convey potent cytoprotective effects hemming the inflammatory mechanisms are expected to provide clinical benefits. In this regard, we review the cytoprotective protease activated protein C (aPC) and its strong anti-inflammatory effects thereby preventing the ensuing thrombotic complications in CVD. Furthermore, restoring GAG-like vasculo-protection, such as providing heparin-proteoglycan mimetics to improve regulation of platelet and coagulation activity and to suppress of endothelial perturbance and leukocyte-derived pro-inflammatory cytokines, may provide a path to alleviate thrombo-inflammatory disorders in the future. The vascular tissue-modeled heparin proteoglycan mimic, antiplatelet and anticoagulant compound (APAC), dual antiplatelet and anticoagulant, is an injury-targeting and locally acting arterial antithrombotic which downplays collagen- and thrombin-induced and complement-induced activation and protects from organ injury.

AFM investigation of APAC (antiplatelet and anticoagulant heparin proteoglycan).

Antiplatelet and anticoagulant drugs are classified antithrombotic agents with the purpose to reduce blood clot formation. For a successful treatment of many known complex cardiovascular diseases driven by platelet and/or coagulation activity, the need of more than one antithrombotic agent is inevitable. However, combining drugs with different mechanisms of action enhances risk of bleeding. Dual anticoagulant and antiplatelet (APAC), a novel semisynthetic antithrombotic molecule, provides both anticoagulant and antiplatelet properties in preclinical studies. APAC is entering clinical studies with this new exciting approach to manage cardiovascular diseases. For a better understanding of the biological function of APAC, comprehensive knowledge of its structure is essential. In this study, atomic force microscopy (AFM) was used to characterize APAC according to its structure and to investigate the molecular interaction of APAC with von Willebrand factor (VWF), since specific binding of APAC to VWF could reduce platelet accumulation at vascular injury sites. By the optimization of drop-casting experiments, we were able to determine the volume of an individual APAC molecule at around 600 nm3, and confirm that APAC forms multimers, especially dimers and trimers under the experimental conditions. By studying the drop-casting behavior of APAC and VWF individually, we depictured their interaction by using an indirect approach. Moreover, in vitro and in vivo conducted experiments in pigs supported the AFM results further. Finally, the successful adsorption of APAC to a flat gold surface was confirmed by using photothermal-induced resonance, whereby attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) served as a reference method.

COVID-19 adenovirus vaccine triggers antibodies against PF4 complexes to activate complement and platelets.

BACKGROUND: Vaccine-induced thrombotic thrombocytopenia (VITT) is a rare coagulation disorder reported after administration of COVID-19 adenovirus-vectored vaccines. VITT is mediated by anti-platelet factor 4 (PF4) antibodies activating platelets through the Fcγ-receptor II (FcγRII), and it is associated with strong fibrin turnover. The complement system is involved in several other immunothrombotic entities, but its impact on VITT is not established. OBJECTIVE: To assess antibodies in interaction with the activation of platelets and complement triggered by VITT. METHODS: Antibodies against adenovirus type 2 hexon protein, ChAdOx1 adenoviral vector-specific IgG and PF4 were analyzed by enzyme immunoassays from VITT patients (n = 5). The EDTA plasma samples of the patients and controls were used to measure both terminal complement complexes (TCC) by ELISA and aggregation of healthy donor platelets. We studied the effects of human immunoglobulin (IVIG) and glycoprotein IIb/IIIa inhibitor (GPIIb/IIIa) on spontaneous and collagen-induced platelet aggregation supplemented with VITT plasma. RESULTS: None of the patients had experienced a COVID-19 infection. Antibody analyses confirmed the immunogenicity of the adenovirus-vectored ChAdOx1 vaccine. Moreover, VITT plasma had anti-PF4 antibodies and elevated TCC levels as a sign of complement activation. In isolated healthy donor platelets, VITT patient plasma caused marked, spontaneous aggregation of platelets, which was abolished by eptifibatide and high-dose therapeutic IVIG. CONCLUSIONS: Our findings suggest that VITT is triggered by antibodies against adenovirus vector and PF4-polyanion complexes which strongly co-activate complement and platelets. The spontaneous platelet aggregation was suppressed by IVIG or eptifibatide, indicating that besides FcγRII, also GPIIb/IIIa receptor exerts platelet procoagulant role in VITT.

Safety and Functional Pharmacokinetic Profile of APAC, a Novel Intravascular Antiplatelet and Anticoagulant.

ABSTRACT: Vascular intervention-induced platelet and coagulation activation is often managed with a combination of antiplatelets and anticoagulants, with evident benefits, but with a risk of systemic bleeding. Antiplatelet and anticoagulant (APAC) is a dual antiplatelet and anticoagulant heparin bioconjugate, which targets vascular injury sites to act as a local antithrombotic. We assessed the nonclinical safety and exposure of intravenously infused APAC in rats and cynomolgus monkeys by using single-day and 14-day repeat dose toxicology and pharmacodynamic markers. Activated partial thromboplastin time (APTT) was used as a functional surrogate of anticoagulant exposure of APAC. Routine clinical in-life observations were followed by clinical pathology and necropsy. The no-observed-adverse-effect level (NOAEL) in rats for the single APAC dose was 20 mg/kg and for the repeated administration was 10 mg/kg/d. Monkeys tolerated a single APAC dose of 10 mg/kg, although the red blood cell count reduced 16%-19% correlating with tissue hemorrhage at vein puncture and affected muscle sites during handling of the animals. However, after 2-week recovery, all clinical signs were normal. The single dose NOAEL exceeded 3 mg/kg. The repeat administration of 3-6 mg/kg/d of APAC was tolerated, but some clinical signs were observed. The NOAEL for repeated dosing was 0.5 mg/kg/d. APAC prolonged APTT dose-dependently in both species, returning to baseline after 1.5 (<10 mg/kg) or essentially by 6 hours also under repetitive dosing. The toxicology profile supports the safety of an intravenous APAC dose of 0.5 mg/kg/d for possible clinical applications. APTT is an acceptable indicator of the immediate systemic anticoagulation effect of APAC.

Platelets compensate for poor thrombin generation in type 3 von Willebrand disease.

In type 3 von Willebrand disease (VWD3), the most severe form with absent von Willebrand factor (VWF), the bleeding phenotype is variable. Platelet contribution to the hemostatic defect in VWD3 calls upon further studies. We investigated the contribution of platelets to in vitro thrombin generation (TG) and platelet procoagulant activity in VWD3. TG was assessed by calibrated automated thrombogram (CAT) in platelet-poor (PPP) and -rich plasma (PRP) from 9 patients before and in 6 patients also 30 min after receiving their regular VWF therapy. Responsiveness of PPP to FVIII and protein S was also investigated. TG data were compared with routine laboratory variables, rotational thromboelastometry (ROTEM) and platelet expression of P-selectin and phosphatidylserine in flow cytometry. Compared with healthy controls, TG was markedly decreased in VWD3 PPP (peak thrombin was 16% of normal median), but not in PRP (77% of normal median) (p = 0.002). Six out of nine patients (67%) were high responders in their platelet P-selectin, and 5/9 (56%) in phosphatidylserine expression. Replacement therapy improved TG in PPP, while in PRP TG only modestly increased or was unaffected. In PPP, FVIII levels associated with TG and in vitro FVIII-supplemented TG inclined up to threefold. Conversely, a FVIII inhibitory antibody reduced plasma TG in all, but especially in patients with remnant FVIII levels. Inhibition of protein S improved plasma TG, particularly at low FVIII levels. ROTEM failed to detect VWD3.In VWD3, TG is reduced in PPP and regulated by FVIII and protein S, but TG is close to normal in PRP. VWD3 platelets seem to compensate for the FVIII-associated reduction in TG by their exposure of P-selectin and phosphatidylserine.

Novel Locally Acting Dual Antiplatelet and Anticoagulant (APAC) Targets Multiple Sites of Vascular Injury in an Experimental Porcine Model.

OBJECTIVES: Vascular binding of dual antiplatelet and anticoagulant (APAC) was assessed in surgically created femoral arteriovenous fistula (AVF) and iliac and carotid artery injury in porcine models. METHODS: Three models of collagen exposing injury were used: 1) femoral AVF, 2) in vivo iliac and carotid artery balloon angioplasty injury, and 3) in vitro femoral artery endothelial denudation injury. Biotinylated APAC (0.5 mg/mL) was incubated with the injury site before releasing blood flow. APAC, von Willebrand factor (vWF), laminin, platelet endothelial cell adhesion molecule 1 (PECAM-1), and podocalyxin were detected in histological sections using immunofluorescence and confocal microscopy and Manders' co-localisation coefficient (M1). RESULTS: APAC bound to AVF at anastomosis and to both in vivo and in vitro injured arteries. APAC co-localised with matrix vWF (M1 ≥ 0.66) and laminin (M1 ≥ 0.60), but less so if endothelial PECAM-1 or podocalyxin was present (M1 ≤ 0.25). APAC targeted and penetrated the injured vessel wall, especially the AVF vein. CONCLUSIONS: APAC, compatible with its high negative charge, rapidly targets injured vessels co-localizing with matrix vWF and laminin, but not with endothelial PECAM-1 and podocalyxin. This localising feature may have potential antithrombotic implications for vascular interventions.

Heparan sulfates are critical regulators of the inhibitory megakaryocyte-platelet receptor G6b-B.

The immunoreceptor tyrosine-based inhibition motif (ITIM)-containing receptor G6b-B is critical for platelet production and activation. Loss of G6b-B results in severe macrothrombocytopenia, myelofibrosis and aberrant platelet function in mice and humans. Using a combination of immunohistochemistry, affinity chromatography and proteomics, we identified the extracellular matrix heparan sulfate (HS) proteoglycan perlecan as a G6b-B binding partner. Subsequent in vitro biochemical studies and a cell-based genetic screen demonstrated that the interaction is specifically mediated by the HS chains of perlecan. Biophysical analysis revealed that heparin forms a high-affinity complex with G6b-B and mediates dimerization. Using platelets from humans and genetically modified mice, we demonstrate that binding of G6b-B to HS and multivalent heparin inhibits platelet and megakaryocyte function by inducing downstream signaling via the tyrosine phosphatases Shp1 and Shp2. Our findings provide novel insights into how G6b-B is regulated and contribute to our understanding of the interaction of megakaryocytes and platelets with glycans.

Gain-of-function CEBPE mutation causes noncanonical autoinflammatory inflammasomopathy.

BACKGROUND: CCAAT enhancer-binding protein epsilon (C/EBPε) is a transcription factor involved in late myeloid lineage differentiation and cellular function. The only previously known disorder linked to C/EBPε is autosomal recessive neutrophil-specific granule deficiency leading to severely impaired neutrophil function and early mortality. OBJECTIVE: The aim of this study was to molecularly characterize the effects of C/EBPε transcription factor Arg219His mutation identified in a Finnish family with previously genetically uncharacterized autoinflammatory and immunodeficiency syndrome. METHODS: Genetic analysis, proteomics, genome-wide transcriptional profiling by means of RNA-sequencing, chromatin immunoprecipitation (ChIP) sequencing, and assessment of the inflammasome function of primary macrophages were performed. RESULTS: Studies revealed a novel mechanism of genome-wide gain-of-function that dysregulated transcription of 464 genes. Mechanisms involved dysregulated noncanonical inflammasome activation caused by decreased association with transcriptional repressors, leading to increased chromatin occupancy and considerable changes in transcriptional activity, including increased expression of NLR family, pyrin domain-containing 3 protein (NLRP3) and constitutively expressed caspase-5 in macrophages. CONCLUSION: We describe a novel autoinflammatory disease with defective neutrophil function caused by a homozygous Arg219His mutation in the transcription factor C/EBPε. Mutated C/EBPε acts as a regulator of both the inflammasome and interferome, and the Arg219His mutation causes the first human monogenic neomorphic and noncanonical inflammasomopathy/immunodeficiency. The mechanism, including widely dysregulated transcription, is likely not unique for C/EBPε. Similar multiomics approaches should also be used in studying other transcription factor-associated diseases.

Evaluation of a standardized protocol for thrombin generation using the calibrated automated thrombogram: A Nordic study.

INTRODUCTION: The thrombin generation assay-calibrated automated thrombogram (TGA-CAT) method is used to measure the overall coagulation capacity in plasma. However, the method is still considered to be a research tool, mainly because of its' lack of standardization. AIM: Our study aimed to further raise the standardization level for the TGA-CAT method by evaluating a detailed standardization protocol and three reference plasmas' (RP)s ability to normalize results. METHODS: Six Nordic centres participated in the study, and with input from all centres a detailed laboratory standardization protocol based on the TGA-CAT manual of the manufacturer was established. Three types of plasma, hypo-,normal and hypercoagulable plasma were assessed. Three commercial lyophilized RPs were used for normalization of data. All samples were aliquoted at the Malmö centre and sent frozen at -20˚C to participating centres. RESULTS: Before normalization, all results under all testing conditions showed inter-laboratory coefficient of variability of 10% or lower except for endogenous thrombin potential (12%) and peak (14%) in hypo-plasma with 1 pmol/L tissue factor as starting agent. Successful normalization, improving variability in results, was obtained with two of the three evaluated RPs (HemosIL RP and Affinity RP). CONCLUSION: With our standardization concept, we were able to produce TGA-CAT results as robust as standard coagulation assays used in the routine laboratories. Normalization with HemosIL RP may be considered in populations with low or unknown coagulability, while when analysing plasma samples from populations where hypercoagulability is known or suspected, normalization with Affinity RP may be preferred.

Dual antiplatelet and anticoagulant (APAC) heparin proteoglycan mimetic with shear-dependent effects on platelet-collagen binding and thrombin generation.

Heparin proteoglycans (HEP-PGs) carry standard heparin-mediated anticoagulant properties as well as novel antiplatelet functions, a combination that may be significant for targeting multiple pathways in a single therapy. Recent work developing semisynthetic HEP-PG mimetics has shown promising results also in vivo, however flow conditions in vitro that replicate in vivo hemodynamics have not been reported. In this work, we present several assays (platelet calcium mobilization, aggregometry, microfluidic tests at venous and arterial hemodynamics) to characterize specific mechanistic effects of dual antiplatelet and anticoagulant (APAC) constructs as mimetics of HEP-PGs. Three APACs with different conjugation levels of heparin chains (CL10, CL18, HICL) were shown to decrease platelet deposition to collagen surfaces in PPACK-treated whole blood at venous shear rate (200 s-1). FXIIa-inhibited whole blood (CTI: corn trypsin inhibitor, 40 µg/mL) perfused over collagen/tissue factor showed reduced both platelet and fibrin deposition when treated with APACs. IC50 values for platelet and fibrin inhibition were calculated for each molecule at venous shear rate. Increasing the shear rate to arterial flows (1000 s-1) and using APAC as the sole anticoagulant, resulted in a more potent antiplatelet effect of APAC, suggesting an added effect on von Willebrand Factor (vWF) function. Additionally, APAC caused an inhibition of calcium mobilization specific to thrombin and collagen stimulation and a dose-dependent reduction in collagen-mediated platelet aggregation. Understanding the sensitivity of APAC activity to shear rate, platelet signaling and procoagulant pathways is important for applications in which APAC administration may have beneficial therapeutic effects.

Dual antiplatelet and anticoagulant APAC prevents experimental ischemia-reperfusion-induced acute kidney injury.

BACKGROUND: Renal ischemia-reperfusion predisposes to acute kidney injury (AKI) and mortality. APAC, mast cell heparin proteoglycan mimetic is a potent dual antiplatelet and anticoagulant inhibiting thrombosis in several vascular models. METHODS: Clinically relevant (0.06 and 0.13 mg/kg) and high (0.32 and 7.3 mg/kg) heparin doses of APAC and unfractionated heparin (UFH) were administered i.v. in pharmacological studies. Antithrombotic action of APAC and UFH was assessed with platelet aggregation to collagen, activated partial thromboplastin (APTT) and prothrombin (PT) times. Pharmacodynamics of [64Cu]-APAC or -UFH were monitored by PET/CT. Next, APAC and UFH doses (0.06 and 0.13 mg/kg) were i.v. administered 10 min prior to renal ischemia-reperfusion injury (IRI) in rats. RESULTS: APAC in contrast to UFH inhibited platelet aggregation. During 0.06 and 0.13 mg/kg dose regimens APTT and PT remained at baseline, but at the high APTT prolonged fourfold to sixfold. Overall bio-distribution and clearance of APAC and UFH were similar. After bilateral 30-min renal artery clamping, creatinine, urea nitrogen and neutrophil gelatinase-associated lipocalin concentrations and histopathology indicated faster renal recovery by APAC (0.13 mg/kg). APAC, unlike UFH, prevented expression of innate immune ligand hyaluronan and tubulointerstitial injury marker Kim-1. Moreover, in severe bilateral 1-h renal artery clamping, APAC (0.13 mg/kg) prevented AKI, as demonstrated both by biomarkers and survival. Compatible with kidney protection APAC reduced the circulating levels of vascular destabilizing and pro-inflammatory angiopoietin-2 and syndecan-1. No tissue bleeding ensued. CONCLUSION: APAC and UFH were similarly eliminated via kidneys and liver. In contrast to UFH, APAC (0.13 mg/kg) was reno-protective in moderate and even severe IRI by attenuating vascular injury and innate immune activation.

Factor XIII deficiency enhances thrombin generation due to impaired fibrin polymerization - An effect corrected by Factor XIII replacement.

INTRODUCTION: Factor XIII (FXIII) cross-links fibrin, completing blood coagulation. Congenital FXIII deficiency is managed with plasma-derived FXIII (pdFXIII) or recombinant FXIII (rFXIII) concentrates. AIM: As the mechanisms protecting patients with low FXIII levels (<5IU/dL) from spontaneous bleeds remain unknown we assessed the interplay between thrombin generation (TG), fibrin formation and clot kinetics before and after FXIII administration in three patients with FXIII deficiency. METHODS: Patients received initially rFXIII (35IU/kg, A-subunit) following with pdFXIII at 1250IU or 2500IU (12-30IU/kg) monthly. TG (CAT), thromboelastometry (ROTEM), prothrombin fragments F1+2, fibrinogen and FXIII activity (FXIII:C) were measured at baseline and one-hour recovery. RESULTS: FXIII was at the target level of 20±6IU/dL at the 4-week trough. rFXIII corrected FXIII to 98±15 and high-dose pdFXIII to a level of 90±6, whereas low-dose/half dose pdFXIII reached 45±4IU/dL. Although fibrinogen (Clauss Method) was normal, coagulation in FIBTEM was impaired, which FXIII administration tended to correct. CAT implied 1.6- to 1.9-fold enhanced TG, which FXIII administration normalized. Inhibition of fibrin polymerization by Gly-Pro-Arg-Pro peptide mimicked FXIII deficiency in CAT by enhancing TG both in control and FXIII recovery plasma. Antithrombin, α2-macroblobulin-thrombin complex and prothrombin were normal, whereas F1+2 were elevated compatible with in vivo TG. DISCUSSION: FXIII deficiency impairs fibrinogen function and fibrin formation simultaneously enhancing TG on the poorly polymerizing fibrin strands, when fibrin's antithrombin I -like function is absent. Our study suggests an inverse link between low FXIII levels and enhanced TG modifying structure-function relationship of fibrin to support hemostasis.

Enhanced thrombin generation and reduced intact protein S in processed solvent detergent plasma.

BACKGROUND: Standardized solvent/detergent (S/D)-treated plasma has been developed as an improved alternative to fresh frozen-plasma (FFP) in the management of severe bleeds. This study aimed at exploring compositional modifications that may influence the general applicability of S/D-treated plasma. MATERIALS AND METHODS: S/D-treated plasma and FFP were compared in procoagulant microparticles and concentration of coagulation factors and inhibitors. Compositional differences were correlated with hemostatic and fibrinolytic characteristics as measured by PT, APTT, thrombin generation and thromboelastography. RESULTS: Procoagulant microparticles were absent in S/D-treated plasma. Procoagulant factors were within the normal range. Antithrombin, TFPI and protein S antigen may be normal or slightly reduced depending on the duration of the S/D-treatment, but S/D-treated plasmas had only 12-14% intact functional protein S. Thrombin generation was subsequently increased, especially at low tissue factor concentration (1 pM). Plasma coagulation times in PT and APTT were normal, but 1.5-fold reduced in thromboelastography at low TF (1 pM). α2-antiplasmin was reduced with a concomitant 3-4 fold shortened clot lysis time measured by thromboelastography in the presence of TF (10 pM) and tissue-type plasminogen activator (0.2µg/ml). Enhanced fibrin degradation could be normalised with tranexamic acid. CONCLUSIONS: S/D-treatment seems to induce a procoagulant phenotype that results from a strongly reduced level of intact single chain protein S. Whether this may correct the apparent hemostatic imbalance as suggested from the increased fibrinolysis remains to be established. Our findings may bear implications in patients with deficiencies of natural anticoagulants. Co-administration of tranexamic acid appears beneficial to control enhanced fibrinolysis.

Mast cell-derived heparin proteoglycans as a model for a local antithrombotic.

Mast cell-derived heparin proteoglycans (HEP-PG) reside in vascular tissue and serve as a local antithrombotic. Heparin, as used clinically, is isolated from its protein backbone from porcine and bovine gut mucosa. The isolated heparin is an anticoagulant; however, when bound to a protein carrier the heparin conjugates will become antiplatelet agents by inhibiting collagen-induced platelet aggregation and procoagulant activity, as distinct from soluble heparin. HEP-PG, whether soluble or immobilized to a surface, inhibit platelet deposition on the collagen surface in flowing blood. Mimics of HEP-PG, can be tailored to molecules carrying both antiplatelet and anticoagulant (APAC) properties. These molecules can target the vascular injury site and take residence there. Inhibition of thrombus growth using these APACs under these conditions has been demonstrated in several animal models. Although efficacious for antiplatelet and anticoagulant effects, the bleeding time is shorter with APACs than with unfractionated heparin, suggestive of beneficial efficacy/safety ratio. These strategies may be utilized in drug development, where many vascular injury-related problems can be tackled locally.

Agonist occupancy is essential for forward trafficking of AMPA receptors.

Regulated trafficking of AMPA receptors to cell surface and to synapses is an important determinant of neuronal excitability. In the present study, we have addressed the role of agonist binding and desensitization in the early trafficking of glutamate receptor-D (GluR-D) AMPA receptors. Analysis of point-mutated GluR-D receptors, via electrophysiology and immunofluorescence, revealed that agonist-binding activity is essential for efficient delivery to cell surface in transfected cell lines and in neurons. Cotransfection with stargazin could fully rescue the surface expression of nonbinding mutant receptors in cell lines, indicating that stargazin is able to interact with and promote exit of AMPA receptors from endoplasmic reticulum (ER) independently of agonist binding. Secretion of separately expressed ligand-binding domain constructs showed a similar dependency of agonist binding to that observed with full-length GluR-D, supporting the idea that glutamate-induced closure of the binding site cleft is registered by ER quality control as a necessary priming step for transport competence. In contrast to agonist binding, the ability of the receptor to undergo desensitization had only a minor influence on trafficking. Our results are consistent with the hypothesis that AMPA receptors are synthesized as intrinsically unstable molecules, which require glutamate binding for structural stability and for transport-competence.

Determinants of antagonist binding at the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor subunit, GluR-D. Role of the conserved arginine 507 and glutamate 727 residues.

Previous structural and mutagenesis studies indicate that the invariant alpha-amino and alpha-carboxyl groups of glutamate receptor agonists are engaged in polar interactions with oppositely charged, conserved arginine and glutamate residues in the ligand-binding domain of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor. To examine the role of these residues (R507 and E727 in the GluR-D subunit) in the discrimination between agonists and antagonists, we analyzed the ligand-binding properties of homomeric GluR-D and its soluble ligand-binding domain with mutations at these positions. Filter-binding assays using [3H]AMPA, an agonist, and [3H]Ro 48-8587, a high-affinity antagonist, as radioligands revealed that even a conservative mutation at R507 (R507K) resulted in the complete loss of both agonist and antagonist binding. In contrast, a negative charge at position 727 was necessary for agonist binding, whereas the isosteric mutation, E727Q, abolished all agonist binding but retained high-affinity binding for [3H]Ro 48-8587, displaceable by 7,8-dinitroquinoxaline-2,3-dione. Competition binding studies with antagonists representing different structural classes in combination with ligand docking experiments suggest that the role of E727 is antagonist-specific, ranging from no interaction to weak electrostatic interactions involving indirect and direct hydrogen bonding with the antagonist molecule. These results underline the importance of ion pair interaction with E727 for agonist activity and suggest that an interaction with R507, but not with E727, is essential for antagonist binding.