Coagulation and platelet biology at the intersection of health and disease: illustrated capsules of the 11th Symposium on Hemostasis at the University of North Carolina.

The University of North Carolina Symposia on Hemostasis began in 2002, with The First Symposium on Hemostasis with a Special Focus on FVIIa and Tissue Factor. They have occurred biannually since and have maintained the primary goal of establishing a forum for the sharing of outstanding advances made in the basic sciences of hemostasis. The 2024 11th Symposium on Hemostasis will bring together leading scientists from around the globe to present and discuss the latest research related to coagulation factors and platelet biology. In keeping with the tradition of the conference, we expect novel cross-disciplinary collaborations to result from bringing together fundamental scientists and physician-scientists from different backgrounds and perspectives. The aim of these collaborations is to springboard the next generation of important advances in the field. This year's program was designed to discuss Coagulation and Platelet Biology at the Intersection of Health and Disease. The goal is to develop a better understanding of the pathophysiologic mechanisms leading to hemostatic and thrombotic disorders as this understanding is critical for the continued development of safe and efficacious therapeutics. Included in this review article are illustrated capsules provided by our speakers that highlight the main conclusions of the invited talks.

Fibrin structure, viscoelasticity and lysis face the interplay of biorelevant polyions.

PURPOSE OF REVIEW: In the past 5 decades, heparins have been widely used as anticoagulants in the prevention and treatment of thrombosis. Subsequent development of heparin variants of various size and charge facilitated the discovery of their multiple biological actions and nonanticoagulant benefits. Platelet-derived or microbial polyphosphates, as well as DNA released in the course of neutrophil extracellular trap-formation are additional polyanions, which can modulate the development and stability of thrombi associated with cancer or inflammation. In this review, we focus on the size-dependent and electric charge-dependent modulatory effects of the three polyanions of different chemical structure. RECENT FINDINGS: The polycationic histones have been recognized as potential biomarkers and therapeutic targets in several diseases related to inflammation and thrombosis. Since combating histones with activated protein C or heparin could cause unwanted bleeding, the quest for nonanticoagulant histone-neutralizing agents is ongoing. Polyanions may neutralize or exaggerate certain histone-mediated effects depending on their electric charge, size and histone effects under investigation. Several prothrombotic effects of polyphosphates and DNA are also size-dependent. SUMMARY: The efficiency of future therapeutics targeting prothrombotic polyanions or histones is not a simple matter of electric charge, but may rely on a delicate combination of size, charge and chemical composition.

Polyphosphate nanoparticles enhance the fibrin stabilization by histones more efficiently than linear polyphosphates.

INTRODUCTION: Beyond the three-dimensional fibrin network, the mechanical and lytic stability of thrombi is supported by the matrix of neutrophil extracellular traps (NETs) composed of polyanionic DNA meshwork with attached proteins including polycationic histones. Polyphosphates represent another type of polyanions, which in their linear form are known to enhance the fibrin stabilizing effects of DNA and histones. However, in vivo polyphosphates are also present in the form of nanoparticles (PolyP-NP), the interference of which with the fibrin/NET matrix is poorly characterized. AIMS: To compare the effects of linear and nanoparticulate polyphosphates, and their combinations with relevant NET components (DNA, histone H3) on fibrin formation, structure, and lysis in in vitro assays focusing on histone-polyphosphate interactions. METHODS: Transmission electron microscopy and dynamic light scattering for stability of the PolyP-NP preparations. Turbidimetry for kinetics of fibrinogen clotting by thrombin and fibrin dissolution by tissue-type plasminogen activator/plasminogen. Scanning electron microscopy for fibrin structure. Surface plasmon resonance for strength of histone-PolyP interactions. RESULTS: Both linear PolyP and PolyP-NP accelerated the fibrin formation and slowed down its dissolution and these effects were strongly dependent on the number of individual PolyP particles and not on their size. Addition of DNA did not modify significantly the PolyP-NP effects on fibrin formation and lysis. Both linear and nanoparticulate PolyP counteracted the effect of histone in the acceleration of fibrinogen clotting by thrombin. PolyP-NP, but not linear PolyP enhanced the prolongation of lysis time in fibrin containing histone and caused more pronounced thickening of the fibrin fibers than the linear form. Finally, PolyP-NP bound weaker to histone than the linear form. CONCLUSIONS: The interaction of PolyP with histone was a stronger modulator of fibrin formation and lysis than its interaction with DNA. In addition, the PolyP nanoparticles enhanced the thrombus stabilizing effects of histone more effectively than linear PolyP.

Cryopreservation moderates the thrombogenicity of arterial allografts during storage.

INTRODUCTION: Management of vascular infections represents a major challenge in vascular surgery. The use of cryopreserved vascular allografts could be a feasible therapeutic option, but the optimal conditions for their production and use are not precisely defined. AIMS: To evaluate the effects of cryopreservation and the duration of storage on the thrombogenicity of femoral artery allografts. METHODS: In our prospective study, eleven multi-organ-donation-harvested human femoral arteries were examined at five time points during storage at -80°C: before cryopreservation as a fresh native sample and immediately, one, twelve and twenty-four weeks after the cryopreservation. Cross-sections of allografts were perfused with heparin-anticoagulated blood at shear-rates relevant to medium-sized arteries. The deposited platelets and fibrin were immunostained. The thrombogenicity of the intima, media and adventitia layers of the artery grafts was assessed quantitatively from the relative area covered by fibrin- and platelet-related fluorescent signal in the confocal micrographs. RESULTS: Regression analysis of the fibrin and platelet coverage in the course of the 24-week storage excluded the possibility for increase in the graft thrombogenicity in the course of time and supported the hypothesis for a descending trend in fibrin generation and platelet deposition on the arterial wall. The fibrin deposition in the cryopreserved samples did not exceed the level detected in any of the three layers of the native graft. However, an early (up to week 12) shift above the native sample level was observed in the platelet adhesion to the media. CONCLUSIONS: The hemostatic potential of cryopreserved arterial allografts was retained, whereas their thrombogenic potential declined during the 6-month storage. The only transient prothrombotic change was observed in the media layer, where the platelet deposition exceeded that of the fresh native grafts in the initial twelve weeks after cryopreservation, suggesting a potential clinical benefit from antiplatelet therapy in this time-window.

Size- and charge-dependent modulation of the lytic susceptibility and mechanical stability of fibrin-histone clots by heparin and polyphosphate variants.

BACKGROUND: Neutrophil extracellular traps (NETs) containing DNA and histones are expelled from neutrophils in infection and thrombosis. Heparins, anticoagulant polyanions, can neutralize histones with a potential therapeutic advantage in sepsis. Polyphosphates, procoagulant polyanions, are released by platelets and microorganisms. OBJECTIVES: To characterize the combined effects of NET components and polyanions on clot structure, mechanical properties and lytic susceptibility. METHODS: Scanning electron microscopy, pressure-driven permeation, turbidimetry, and oscillation rheometry were used for the characterization of the structure, viscoelasticity, and kinetics of formation and lysis of fibrin and plasma clots containing histones+/-DNA in combination with unfractionated heparin, its desulfated derivatives, low molecular weight heparin (LMWH), pentasaccharide, and polyphosphates of different sizes. RESULTS: Histones and DNA inhibited fibrin lysis by plasmin, but this behavior was not neutralized by negatively charged heparins or short polyphosphates. Rather, fibrin lysis was further inhibited by added polyanions. Histones inhibited plasma clot lysis by tissue plasminogen activator and the response to added heparin was size dependent. Unfractionated heparin, LMWH, and pentasaccharide had no effect, exacerbated, or reversed histone inhibition, respectively. Histones increased the mechanical strength of fibrin, which was exacerbated by smaller heparin and polyphosphate molecules. Histones increased fibrin diameter and pore size of fibrin clots and this effect was neutralized by all heparin variants but enhanced by polyphosphates. CONCLUSIONS: Despite their common polyanionic character, heparins and polyphosphates exert distinct effects on fibrin mechanical and fibrinolytic stability. Anti-fibrinolytic effects of histones were more often enhanced by polyanions not counteracted. Careful selection of anti-histone strategies is required if they are to be combined with thrombolytic therapy.

Biorelevant polyanions stabilize fibrin against mechanical and proteolytic decomposition: Effects of polymer size and electric charge.

The release of neutrophil extracellular traps (NETs) containing DNA and histones is an essential mechanism in the neutrophil-mediated innate immunity. In thrombi the polyanionic DNA confers mechanical and lytic resistance to fibrin and heparins interfere with the effects of NET components. Heparins are polyanions used not only as therapeutic agents, but they are also released by mast cells at entry sites of pathogens. Platelets and microorganisms release a different type of polyanions (polyphosphates) of various size (in the range 60-1000 phosphate monomers). With the current study we aimed to evaluate if the stability of fibrin is influenced by the type of polyanion, its molecular size or relative electric charge. Fibrin structure was approached with scanning electron microscopy (SEM) and pressure-driven permeation. An oscillation rheometer was used to investigate viscoelastic properties. Kinetic turbidimetric assays for the generation and dissolution of composite fibrin clots containing unfractionated heparin (UFH), and its partially or fully desulfated derivatives, as well as low molecular-weight heparin (LMWH), pentasaccharide (S5), and polyphosphates composed of 45 (P45), 100 (P100) or 700 (P700) monomers at average. The smaller polyanions P45, P100, LMWH, and S5 accelerated, whereas P700 and UFH retarded clot formation. All polyanions altered the fibrin structure: SEM and clot permeation showed thicker fibers with smaller (LMWH, S5, P700) or larger (UFH, P100) pores. All polyanions stabilized the clots mechanically, but the smaller P45, P100 and LMWH decreased the deformability of fibrin, whereas the large UFH and P700 increased the maximal bearable deformation of clots. Despite the size-dependent structural changes, all heparins caused a 10-15% prolongation of lysis-times with plasmin, and UFH-effects depended on sulfation patterns. The 20-35% prolongation of lysis-times caused by all polyphosphates was a kringle-dependent phenomenon, and was dampened in the presence of 6-aminohexanoate blocking the lysine-binding sites of plasmin. In summary, we found that polyanions of different chemical structure stabilize fibrin clots via size-dependent modulation of fibrin structure and kringle-dependent inhibition of plasmin-mediated fibrinolysis.

Neutrophils and neutrophil extracellular traps enhance venous thrombosis in mice bearing human pancreatic tumors.

Pancreatic cancer is associated with a high incidence of venous thromboembolism. Neutrophils have been shown to contribute to thrombosis in part by releasing neutrophil extracellular traps (NET). A recent study showed that increased plasma levels of the NET biomarker, citrullinated histone H3 (H3Cit), are associated with venous thromboembolism in patients with pancreatic and lung cancer but not in those with other types of cancer, including breast cancer. In this study, we examined the contribution of neutrophils and NET to venous thrombosis in nude mice bearing human pancreatic tumors. We found that tumor-bearing mice had increased circulating neutrophil counts and levels of granulocyte-colony stimulating factor, neutrophil elastase, H3Cit and cell-free DNA compared with controls. In addition, thrombi from tumor-bearing mice contained increased levels of the neutrophil marker Ly6G, as well as higher levels of H3Cit and cell-free DNA. Thrombi from tumor-bearing mice also had denser fibrin with thinner fibers consistent with increased thrombin generation. Importantly, either neutrophil depletion or administration of DNase I reduced the thrombus size in tumor-bearing but not in control mice. Our results, together with clinical data, suggest that neutrophils and NET contribute to venous thrombosis in patients with pancreatic cancer.

Functional cyclophilin D moderates platelet adhesion, but enhances the lytic resistance of fibrin.

In the course of thrombosis, platelets are exposed to a variety of activating stimuli classified as 'strong' (e.g. thrombin and collagen) or 'mild' (e.g. ADP). In response, activated platelets adhere to injured vasculature, aggregate, and stabilise the three-dimensional fibrin scaffold of the expanding thrombus. Since 'strong' stimuli also induce opening of the mitochondrial permeability transition pore (MPTP) in platelets, the MPTP-enhancer Cyclophilin D (CypD) has been suggested as a critical pharmacological target to influence thrombosis. However, it is poorly understood what role CypD plays in the platelet response to 'mild' stimuli which act independently of MPTP. Furthermore, it is unknown how CypD influences platelet-driven clot stabilisation against enzymatic breakdown (fibrinolysis). Here we show that treatment of human platelets with Cyclosporine A (a cyclophilin-inhibitor) boosts ADP-induced adhesion and aggregation, while genetic ablation of CypD in murine platelets enhances adhesion but not aggregation. We also report that platelets lacking CypD preserve their integrity in a fibrin environment, and lose their ability to render clots resistant against fibrinolysis. Our results indicate that CypD has opposing haemostatic roles depending on the stimulus and stage of platelet activation, warranting a careful design of any antithrombotic strategy targeting CypD.

Arginase activity, urea, and hydroxyproline concentration are reduced in keratoconus keratocytes.

PURPOSE: Keratoconus (KC) is a disease characterized by thinning and deformation of the cornea, but its etiology remains unknown. Seventy percent of the corneal stroma consists of collagen, which is composed of three intertwined polypeptide chains with glycine-hydroxyproline-proline repeats along their sequence. Arginase is a cytoplasmatic enzyme and catalyzes the conversion of arginine to urea and ornithine, which serves as a precursor for the endogenous synthesis of proline and hydroxyproline. The purpose of this study was to analyze arginase activity, as well as collagen and urea formation in normal and KC-keratocytes and to determine the impact of urea on keratocyte viability and proliferation in vitro. METHODS: Primary human keratocytes were isolated by digestion in collagenase (1.0 mg/mL) from surgically removed corneas of eight keratoconus patients and eight normal human corneal buttons and cultured in DMEM/Ham's F12 medium supplemented with 5 % fetal calf serum. Arginase activity and urea concentration were measured in cell-lysates, hydroxyproline concentration in supernatant of cultured keratocytes using colorimetric assay. Cell viability and cell proliferation of cultured keratocytes were assessed after treatment with urea at concentrations up to10 mM for 24 h using assays for metabolic activity and DNA replication. RESULTS: Arginase activity and urea concentration in KC-keratocytes decreased by about 50 % compared to normal keratocytes (p = 0.003 and p = 0.008). Hydroxyproline synthesized by cultured KC-keratocytes was also approximately 50 % less compared to normal keratocytes (p = 0.02) and this difference decreased following treatment with 5.0 or 10.0 mM urea (p = 0.02; 0.03), without any change in cell viability (p > 0.09). However, the urea treatment increased modestly (by 20 %) the proliferation rate of KC-keratocytes (p = 0.04; 0.04; 0.04), without any effect on normal cultured keratocytes (p > 0.09). CONCLUSIONS: We identified suppressed arginase activity in the metabolic program of cultured keratoconus keratocytes. The level of urea, as one product of the enzyme arginase was also decreased. This results in impaired collagen synthesis, evidenced in the culture by reduced hydroxyproline concentration. In addition, our data showed that the other product of the arginase reaction, urea supports the proliferation of KC-keratocytes, without changes in their viability. The metabolic reprogramming of keratoconus keratocytes and its impact on development of a clinically detectable keratoconus disease has to be further analyzed.

Bleeding related to disturbed fibrinolysis.

The components and reactions of the fibrinolysis system are well understood. The pathway has fewer reactants and interactions than coagulation, but the generation of a complete quantitative model is complicated by the need to work at the solid-liquid interface of fibrin. Diagnostic tools to detect disease states due to malfunctions in the fibrinolysis pathway are also not so well developed as is the case with coagulation. However, there are clearly a number of inherited or acquired pathologies where hyperfibrinolysis is a serious, potentially life-threatening problem and a number of antifibrinolytc drugs are available to treat hyperfibrinolysis. These topics will be covered in the following review.

Femtosecond laser-assisted cataract surgery in Alport syndrome with anterior lenticonus.

PURPOSE: To report the surgical treatment of 3 eyes of 2 patients with bilateral anterior lenticonus due to Alport syndrome using femtosecond laser-assisted cataract surgery (FLACS). METHODS: Two patients with Alport syndrome presented to our department due to anterior lenticonus in both eyes. We performed FLACS with posterior chamber lens implantation in both eyes of one patient and in one eye of the other patient. Anterior segment morphologic changes were visualized with a Scheimpflug camera, and anterior segment optical coherence tomography preoperatively and 3 months after surgery. Ultrastructure of the cut capsule edges was observed with scanning electron microscopy and compared to the edge of femtosecond laser capsulotomy performed on an otherwise healthy patient with cataract (control). RESULTS: The intraocular lens (IOL) postoperative positioning parameters met the international requirements of aspherical and wavefront customized IOLs (tilt <10 degree, decentration <800 µm). Scanning electron microscopy revealed the same characteristics of the cut capsule edges in the Alport and in the control eyes. CONCLUSIONS: Femtosecond laser cataract surgery can be a safe and successful method for optical rehabilitation of anterior lenticonus in patients with Alport syndrome.

Evaluation of the mechanical properties of the anterior lens capsule following femtosecond laser capsulotomy at different pulse energy settings.

PURPOSE: To evaluate and compare the mechanical properties of anterior capsule opening performed with femtosecond laser capsulotomy at different energy settings in ex vivo porcine anterior lens capsule specimens. METHODS: Twenty-five fresh porcine eyes per group were included in the study. Femtosecond laser capsulotomy was performed with three different pulse energy levels: 2 µJ (low energy group), 5 µJ (intermediate energy group), and 10 µJ (high energy group). The capsule openings were stretched with universal testing equipment until they ruptured. The morphologic profile of the cut capsule edges was evaluated using scanning electron microscopy. RESULTS: The high energy group had significantly lower rupture force (108 ± 14 mN) compared to the intermediate energy group (118 ± 10 mN) (P < .05) and low energy group (119 ± 11 mN) (P < .05), but the difference between the intermediate energy and low energy groups was not significant (P = .9479). The high energy group had significantly lower circumference stretching ratio (144% ± 3%) compared to the intermediate energy group (148% ± 3%) (P < .05) and low energy group (148% ± 3%) (P < .05), but the difference between the intermediate energy group and low energy group was not significant (P = .9985). Scanning electron microscopy images showed that the edge was only serrated with low and intermediate energy, but additional signs of collagen melting and denaturation were observed at high energy. CONCLUSIONS: Anterior capsule openings created at a high energy level were slightly weaker and less extensible than those created at low or intermediate levels, possibly due to the increased thermal effect of photo-disruption.

Ultrastructure and composition of thrombi in coronary and peripheral artery disease: correlations with clinical and laboratory findings.

INTRODUCTION: Fibrin structure and cellular composition of thrombi profoundly affect the clinical outcomes in ischemic coronary and peripheral artery disease. Our study addressed the interrelations of structural features of thrombi and routinely measured laboratory parameters. MATERIALS AND METHODS: Thrombi removed by thromboaspiration following acute myocardial infarction (n=101) or thrombendarterectomy of peripheral arteries (n=50) were processed by scanning electron microscopy and immunostaining for fibrin and platelet antigen GPIIb/IIIa to determine fibrin fibre diameter and relative occupancy by fibrin and cells. Correlations between the structural characteristics and selected clinical parameters (age, sex, vascular localization, blood cell counts, ECG findings, antiplatelet medication, accompanying diseases, smoking) were assessed. RESULTS: We observed significant differences in mean fibre diameter (122 vs. 135 nm), fibrin content (70.5% vs. 83.9%), fluorescent fibrin/platelet coverage ratio (0.18 vs. 1.06) between coronary and peripheral thrombi. Coronary thrombi from smokers contained more fibrin than non-smokers (78.1% vs. 62.2% mean occupancy). In the initial 24 h, fibrin content of coronary thrombi decreased with time, whereas in peripheral thrombi platelet content increased in the first 7 days. In coronaries, higher platelet content and smaller vessel diameter were associated with thinner fibrin fibres, whereas hematocrit higher than 0.35 correlated with larger intrathrombotic platelet occupancy. Smoking and dyslipidaemia strengthened the dependence of clot platelet content on systemic platelet count (the adjusted determination coefficient increased from 0.33 to 0.43 and 0.65, respectively). CONCLUSION: Easily accessible clinical parameters could be identified as significant determinants of ultrastructure and composition of coronary and peripheral thrombi.

Comparison of the mechanical properties of the anterior lens capsule following manual capsulorhexis and femtosecond laser capsulotomy.

PURPOSE: To evaluate and compare the mechanical properties of anterior capsule openings performed with the continuous curvilinear capsulorhexis (CCC) technique and femtosecond laser capsulotomy (FLC) in ex vivo porcine lens capsule specimens. METHODS: Fresh porcine eyes were included in the study (CCC group, n = 50; FLC group, n = 30). The capsule openings were stretched with universal testing equipment until they ruptured. The rupture force and circumference stretching ratio were evaluated. The morphologic profile of the cut capsule edges was evaluated using scanning electron microscopy (SEM). RESULTS: The average rupture force was higher in the CCC group (median: 155 mN; interquartile range [IQR]: 129 to 201 mN; range: 71 to 294 mN) than in the FLC group (median: 119 mN; IQR: 108 to 128 mN; range: 91 to 142 mN) (P < .01, Mann-Whitney U test). The average circumference stretching ratio in the CCC group was greater (median: 150%; IQR: 146% to 156%; range: 136% to 161%) than in the FLC group (median: 148%; IQR: 145% to 150%; range: 141% to 154%) (P = .0468, Mann-Whitney U test). When less than 71 mN, no capsular tear occurred in either group. When less than 91 mN, no capsular tear occurred in the FLC group, whereas at 91 mN, the probability of capsular tears was 9% for the CCC group. SEM examination found that the CCC group had smooth edges, whereas those of the FLC group were gently serrated. CONCLUSIONS: According to the current results in a porcine eye model, FLC had less average resistance to capsule tear than CCC, but the weakest openings were seen in the CCC group.

Lytic resistance of fibrin containing red blood cells.

OBJECTIVE: Arterial thrombi contain variable amounts of red blood cells (RBCs), which interact with fibrinogen through an eptifibatide-sensitive receptor and modify the structure of fibrin. In this study, we evaluated the modulator role of RBCs in the lytic susceptibility of fibrin. METHODS AND RESULTS: If fibrin is formed at increasing RBC counts, scanning electron microscopy evidenced a decrease in fiber diameter from 150 to 96 nm at 40% (v/v) RBCs, an effect susceptible to eptifibatide inhibition (restoring 140 nm diameter). RBCs prolonged the lysis time in a homogeneous-phase fibrinolytic assay with tissue plasminogen activator (tPA) by up to 22.7±1.6%, but not in the presence of eptifibatide. Confocal laser microscopy using green fluorescent protein-labeled tPA and orange fluorescent fibrin showed that 20% to 40% (v/v) RBCs significantly slowed down the dissolution of the clots. The fluorescent tPA variant did not accumulate on the surface of fibrin containing RBCs at any cell count above 10%. The presence of RBCs in the clot suppressed the tPA-induced plasminogen activation, resulting in 45% less plasmin generated after 30 minutes of activation at 40% (v/v) RBCs. CONCLUSIONS: RBCs confer lytic resistance to fibrin resulting from modified fibrin structure and impaired plasminogen activation through a mechanism that involves eptifibatide-sensitive fibrinogen-RBC interactions.

Myosin: a noncovalent stabilizer of fibrin in the process of clot dissolution.

Myosin modulates the fibrinolytic process as a cofactor of the tissue plasminogen activator and as a substrate of plasmin. We report now that myosin is present in arterial thrombi and it forms reversible noncovalent complexes with fibrinogen and fibrin with equilibrium dissociation constants in the micromolar range (1.70 and 0.94 microM, respectively). Competition studies using a peptide inhibitor of fibrin polymerization (glycl-prolyl-arginyl-proline [GPRP]) indicate that myosin interacts with domains common in fibrinogen and fibrin and this interaction is independent of the GPRP-binding polymerization site in the fibrinogen molecule. An association rate constant of 1.81 x 10(2) M(-1) x s(-1) and a dissociation rate constant of 3.07 x 10(-4) s(-1) are determined for the fibrinogen-myosin interaction. Surface plasmon resonance studies indicate that fibrin serves as a matrix core for myosin aggregation. The fibrin clots equilibrated with myosin are stabilized against dissolution initiated by plasminogen and tissue-type plasminogen activator (tPA) or urokinase (at fibrin monomer-myosin molar ratio as high as 30) and by plasmin under static and flow conditions (at fibrin monomer-myosin molar ratio lower than 15). Myosin exerts similar effects on the tPA-induced dissolution of blood plasma clots. Covalent modification involving factor XIIIa does not contribute to this stabilizing effect; myosin is not covalently attached to the clot by the time of complete cross-linking of fibrin. Thus, our in vitro data suggest that myosin detected in arterial thrombi binds to the polymerized fibrin, in the bound form its tPA-cofactor properties are masked, and the myosin fibrin clot is relatively resistant to plasmin.