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Non-compressible intra-abdominal hemorrhage (NCIAH) is a major cause of preventable death on the battlefield and in civilian trauma. Currently, it can only be definitively managed with surgery, as there are limited strategies for controlling ongoing NCIAH in the prehospital environment. We hypothesized that a self-propelling thrombin-containing powder (SPTP) could increase survival in a swine model of NCIAH when delivered percutaneously into the closed abdomen using an engineered spray system.Nineteen swine underwent surgical laparotomy followed by a Grade V liver injury that created massive hemorrhage, before closing the abdomen with sutures. Animals either received treatment with standard of care fluid resuscitation (n=9) or the SPTP spray system (n=10), which consisted of a spray device and a 14 Fr catheter. Using the spray system, SPTP was delivered into a hemoperitoneum identified using a focused assessment with sonography in trauma (FAST) exam. Lactated Ringer's solution was administered to all animals to maintain a mean arterial pressure (MAP) of50 mmHg. The primary outcome was percentage of animals surviving at three hours following injury.In the swine model of NCIAH, a greater percentage of animals receiving SPTP survived to three hours, although differences were not significant. The SPTP spray system increased the median survival of animals from 1.6 hr in the fluid resuscitation group to 4.3 hr. The SPTP spray system delivered a total mass of 18.5 ± 1.0 g of SPTP. The mean change in intra-abdominal pressure following SPTP delivery was 5.2 ± 1.8 mmHg (mean ± SEM). The intervention time was 6.7 ± 1.7 min. No adverse effects related to the SPTP formulation or the spray system were observed. SPTP was especially beneficial in animals that had either severely elevated lactate concentrations or low mean arterial pressure of35 mmHg shortly after injury.This demonstrates proof-of-concept for use of a new minimally invasive procedure for managing NCIAH, which could extend survival time to enable patients to reach definitive surgical care.

BackgroundHypercholesterolemia (HC) has previously been shown to augment restenotic response in several animal models and humans. However, the mechanistic aspects of in-stent restenosis (ISR) on a hypercholesterolemic background, including potential augmentation of systemic and local inflammation precipitated by HC are not completely understood. CD47 is a transmembrane protein known to abort crucial inflammatory pathways. Our present studies have examined the interrelation between HC, inflammation, and ISR and investigated the therapeutic potential of stents coated with a CD47-derived peptide (pepCD47) in the hypercholesterolemic rabbit model.Methods and ResultsPepCD47 was immobilized on metal foil coupons and stents using polybisphosphonate coordination chemistry and pyridyldithio/thiol conjugation. The relative abundance of the surface-associated cells on bare metal (BM) and pepCD47 foils exposed to whole rabbit blood showed a 40% inhibition of cell attachment on pepCD47-modified surfaces. Likewise, cytokine expression analyzed in buffy coat-derived cells cultured over the BM and pepCD47-derivatized foils demonstrated a M2/M1 increase with pepCD47 coating. Hypercholesterolemic and normocholesterolemic rabbit cohorts underwent bilateral implantation of BM and pepCD47 stents in the iliac location. Hypercholesterolemia increased neointimal growth in comparison with normocholesterolemic animals at 4 weeks post-stenting. These untoward outcomes were mitigated in the arteries of hypercholesterolemic rabbits treated with pepCD47-derivatized stents. Compared to NC animals, inflammatory cytokine immunopositivity and macrophage infiltration of peri-strut areas increased in HC group animals, and was attenuated in the arteries of hypercholesterolemic rabbits treated with pepCD47 stents.ConclusionsAugmented inflammatory responses triggered by HC underlie severe ISR morphology in hypercholesterolemic rabbits. Blockage of initial platelet and leukocyte attachment to stent struts through CD47 functionalization of stents mitigates pro-restenotic effects of HC.

Background Infection by SARS‐CoV‐2 produces significant pulmonary pathology including endothelial damage with resultant thrombotic events. While pathologic features were described, there are limited data on the relationship of these changes to the inflammatory response and the production of thromboses. Objective To investigate pathology of COVID‐19‐related immunothrombosis. Patients/Methods Tissue samples from lung, kidney, brain and heart that were collected from 45 patients who died of COVID‐19. Histopathological examination was performed after H&E and Picro‐Mallory staining in combination with (immuno)fluorescence to visualize neutrophil extracellular traps. Ultrastructural alterations in lungs were studied with scanning and transmission electron microscopy. Results Inflammatory changes and thrombosis were substantially more pronounced in the lung than in the kidney, heart, and brain. The most common pathologic finding was diffuse alveolar damage. In addition, most lung samples showed thrombi in vessels. The cause of death in single cases was massive pulmonary embolism. Ultrastructural examination revealed neutrophils attached to endothelium, perhaps as a step towards transendothelial migration. In addition, platelets were identified in the midst of fibrin as individual procoagulant balloon‐like cells. Ultrastructural examination demonstrated numerous virion‐like particles. Conclusions Studying (ultra)structural features of the autopsy lung samples from patients with COVID‐19 has provided evidence for a pathogenic link between inflammation and thrombosis. The major features in the lungs of COVID‐19 patients comprised primary inflammatory thrombosis associated with diffuse alveolar damage. The lungs had pronounced circulatory changes with inflammation‐dependent intravascular blood clotting, whereas heart, brain, and kidneys had predominantly degenerative changes that were distinct from the lung pathology.

BACKGROUND: Blood clot contraction, volume shrinkage of the clot, is driven by platelet contraction and accompanied by compaction of the erythrocytes and their gradual shape change from biconcave to polyhedral, with the resulting cells named polyhedrocytes. OBJECTIVES: Here, we examined the role of erythrocyte rigidity on clot contraction and erythrocyte shape transformation. METHODS: We used an optical tracking methodology that allowed us to quantify changes in contracting clot size over time. RESULTS AND CONCLUSIONS: Erythrocyte rigidity has been shown to be increased in sickle cell disease (SCD), and in our experiments erythrocytes from SCD patients were 4-fold stiffer than those from healthy subjects. On average, the final extent of clot contraction was reduced by 53% in the clots from the blood of patients with SCD compared to healthy individuals, and there was significantly less polyhedrocyte formation. To test if this reduction in clot contraction was due to the increase in erythrocyte rigidity, we used stiffening of erythrocytes via chemical cross-linking (glutaraldehyde), rigidifying Wright(b) antibodies (Wr(b)), and naturally more rigid llama ovalocytes. Results revealed that stiffening erythrocytes result in impaired clot contraction and fewer polyhedrocytes. These results demonstrate the role of erythrocyte rigidity in the contraction of blood clots and suggest that the impaired clot contraction/shrinkage in SCD is due to the reduced erythrocyte deformability, which may be an underappreciated mechanism that aggravates obstructiveness of erythrocyte- rich (micro)thrombi in SCD.

Background and Purpose: The purpose was to assess quantitatively and qualitatively the composition and structure of cerebral thrombi and correlate them with the signs of intravital clot contraction (retraction), as well as with etiology, severity, duration, and outcomes of acute ischemic stroke. Methods: We quantified high-resolution scanning electron micrographs of 41 cerebral thrombi for their detailed cellular and noncellular composition and analyzed histological images for the overall structure with the emphasis on red blood cell compression, fibrin age, and the signs of inflammation. Results: Cerebral thrombi were quite compact and had extremely low porosity. The prevailing cell type was polyhedral compressed erythrocytes (polyhedrocytes) in the core, and fibrin-platelet aggregates were concentrated at the periphery; both findings are indicative of intravital contraction of the thrombi. The content of polyhedrocytes directly correlated with the stroke severity. The prevalence of fibrin bundles was typical for more severe cases, while the content of fibrin sponge prevailed in cases with a more favorable course. The overall platelet content in cerebral thrombi was surprisingly small, while the higher content of platelet aggregates was a marker of stroke severity. Fibrillar types of fibrin prevailed in atherothrombogenic thrombi. Older fibrin prevailed in thrombi from the patients who received thrombolytics, and younger fibrin dominated in cardioembolic thrombi. Alternating layers of erythrocytes and fibrin mixed with platelets were common for thrombi from the patients with more favorable outcomes. Thrombi with a higher number of leukocytes were associated with fatal cases. Conclusions: Most cerebral thrombi undergo intravital clot contraction (retraction) that may be of underestimated clinical importance. Despite the high variability of the composition and structure of cerebral thrombi, the content of certain types of blood cells and fibrin structures combined with the morphological signs of intravital contraction correlate with the clinical course and outcomes of acute ischemic stroke.

Bioprosthetic heart valves (BHV) fabricated from glutaraldehyde-fixed heterograft tissue, such as bovine pericardium (BP), are widely used for treating heart valve disease, a group of disorders that affects millions. Structural valve degeneration (SVD) of BHV due to both calcification and the accumulation of advanced glycation end products (AGE) with associated serum proteins limits durability. We hypothesized that BP modified with poly-2-methyl-2-oxazoline (POZ) to inhibit protein entry would demonstrate reduced accumulation of AGE and serum proteins, mitigating SVD. In vitro studies of POZ-modified BP demonstrated reduced accumulation of serum albumin and AGE. BP-POZ in vitro maintained collagen microarchitecture per two-photon microscopy despite AGE incubation, and in cell culture studies was associated with no change in tumor necrosis factor-α after exposure to AGE and activated macrophages. Comparing POZ and polyethylene glycol (PEG)-modified BP in vitro, BP-POZ was minimally affected by oxidative conditions, whereas BP-PEG was susceptible to oxidative deterioration. In juvenile rat subdermal implants, BP-POZ demonstrated reduced AGE formation and serum albumin infiltration, while calcification was not inhibited. However, BP-POZ rat subdermal implants with ethanol pretreatment demonstrated inhibition of both AGE accumulation and calcification. Ex vivo laminar flow studies with human blood demonstrated BP-POZ enhanced thromboresistance with reduced white blood cell accumulation. We conclude that SVD associated with AGE and serum protein accumulation can be mitigated through POZ functionalization that both enhances biocompatibility and facilitates ethanol pretreatment inhibition of BP calcification.

To reveal if coagulopathies relate to the course of COVID-19, we examined 255 patients with moderate and severe COVID-19, receiving anticoagulants and immunosuppressive drugs. Coagulopathy manifested predominantly as hypercoagulability that correlated directly with systemic inflammation, disease severity, comorbidities, and mortality risk. The prolonged clotting tests in about ¼ of cases were associated with high levels of C-reactive protein and antiphospholipid antibodies, which impeded coagulation in vitro. Contraction of blood clots was hindered in about ½ of patients, especially in severe and fatal cases, and correlated directly with prothrombotic parameters. A decrease in platelet contractility was due to moderate thrombocytopenia in combination with platelet dysfunction. Clots with impaired contraction were porous, had a low content of compressed polyhedral erythrocytes (polyhedrocytes) and an even distribution of fibrin, suggesting that the uncompacted intravital clots are more obstructive but patients could also be prone to bleeding. The absence of consumption coagulopathy suggests the predominance of local and/or regional microthrombosis rather than disseminated intravascular coagulation. The results obtained (i) confirm the importance of hemostatic disorders in COVID-19 and their relation to systemic inflammation; (ii) justify monitoring of hemostasis, including the kinetics of blood clot contraction; (iii) substantiate the active prophylaxis of thrombotic complications in COVID-19.

Inflammation and thrombosis are integrated, mutually reinforcing processes, but the interregulatory mechanisms are incompletely defined. Here, we examined the contribution of α-defensins (α-defs), antimicrobial proteins released from activated human neutrophils, on clot formation in vitro and in vivo. Activation of the intrinsic pathway of coagulation stimulates release of α-defs from neutrophils. α-Defs accelerate fibrin polymerization, increase fiber density and branching, incorporate into nascent fibrin clots, and impede fibrinolysis in vitro. Transgenic mice (Def++) expressing human α-Def-1 developed larger, occlusive, neutrophil-rich clots after partial inferior vena cava (IVC) ligation than those that formed in wild-type (WT) mice. IVC thrombi extracted from Def++ mice were composed of a fibrin meshwork that was denser and contained a higher proportion of tightly packed compressed polyhedral erythrocytes than those that developed in WT mice. Def++ mice were resistant to thromboprophylaxis with heparin. Inhibiting activation of the intrinsic pathway of coagulation, bone marrow transplantation from WT mice or provision of colchicine to Def++ mice to inhibit neutrophil degranulation decreased plasma levels of α-defs, caused a phenotypic reversion characterized by smaller thrombi comparable to those formed in WT mice, and restored responsiveness to heparin. These data identify α-defs as a potentially important and tractable link between innate immunity and thrombosis.

Contraction (retraction) of the blood clot is a part of the clotting process driven by activated platelets attached to fibrin that can potentially modulate the obstructiveness and integrity of thrombi. The aim of this work was to reveal the pathogenic importance of contraction of clots and thrombi in venous thromboembolism (VTE). We investigated the kinetics of clot contraction in the blood of 55 patients with VTE. In addition, we studied the ultrastructure of ex vivo venous thrombi as well as the morphology and functionality of isolated platelets. Thrombi from VTE patients contained compressed polyhedral erythrocytes, a marker for clot contraction in vivo. The extent and rate of contraction were reduced by twofold in clots from the blood of VTE patients compared with healthy controls. The contraction of clots from the blood of patients with pulmonary embolism was significantly impaired compared with that of those with isolated venous thrombosis, suggesting that less compacted thrombi are prone to embolization. The reduced ability of clots to contract correlated with continuous platelet activation followed by their partial refractoriness. Morphologically, 75% of platelets from VTE patients were spontaneously activated (with filopodia) compared with only 21% from healthy controls. At the same time, platelets from VTE patients showed a 1.4-fold reduction in activation markers expressed in response to chemical activation when compared with healthy individuals. The results obtained suggest that the impaired contraction of thrombi is an underappreciated pathogenic mechanism in VTE that may regulate the obstructiveness and embologenicity of venous thrombi.

The goal of the study was to establish whether contraction (retraction) of thrombi and/or thrombotic emboli occurs in vivo using morphological signs of blood clot compression, such as characteristic deformation of erythrocytes and redistribution of fibrin toward the surface of a thrombus/embolus. Three postmortem human pulmonary thrombotic emboli were examined by scanning electron microscopy and light microscopy after staining with hematoxylin and eosin as well as with Mallory’s stain. In two pulmonary emboli, which were extracted at 7 and 15 h after death of the patients, compressed polyhedral erythrocytes (polyhedrocytes) were revealed that were formed due to mechanical deformation under the action of contractile forces generated by activated platelets. In addition, the uneven distribution of fibrin within the emboli was found with accumulation of fibrin at the periphery of the emboli, which is another structural characteristic of a contracted blood clot. In one of the three emboli analyzed, which was extracted 38 h after the patient’s death (the “oldest” embolus), the morphological signs of contraction were absent, which was likely related to the partial postmortem autolysis of the embolus or intravital impairment of contraction. The ex vivo thrombotic emboli have morphological signs of clot contraction, suggesting intravital compression of the primary thrombi and/or thrombotic emboli. The in vivo contraction of thrombi and emboli may be an important pathogenic mechanism for modulation of blood flow past otherwise obstructive clots at the sites of thrombotic occlusion of a vessel. The presence of compressed erythrocytes inside and the predominant location of fibrin around the periphery of a thrombus or embolus can potentially serve as additional pathomorphological criteria for the intravital contraction of thrombi and thrombotic emboli.

In-stent restenosis remains an important clinical problem in the era of drug eluting stents. Development of clinical gene therapy protocols for the prevention and treatment of in-stent restenosis is hampered by the lack of adequate local delivery systems. Herein we describe a novel stent-based gene delivery platform capable of providing local arterial gene transfer with adeno-associated viral (AAV) vectors. This system exploits the natural affinity of protein G (PrG) to bind to the Fc region of mammalian IgG, making PrG a universal adaptor for surface immobilization of vector-capturing antibodies (Ab). Our results: 1) demonstrate the feasibility of reversible immobilization of AAV2 vectors using vector tethering by AAV2-specific Ab appended to the stent surface through covalently attached PrG, 2) show sustained release kinetics of PrG/Ab-immobilized AAV2 vector particles into simulated physiological medium in vitro and site-specific transduction of cultured cells, 3) provide evidence of long-term (12 weeks) arterial expression of luciferase with PrG/Ab-tethered AAV2Luc, and 4) show anti-proliferative activity and anti-restenotic efficacy of stent-immobilized AAV2iNOS in the rat carotid artery model of stent angioplasty.

International audience; Background and aimIt was hypothesized that the pattern of coronary occlusion (thrombus composition) might contribute to the onset of ventricular arrhythmia and sudden cardiac death (SCD) in myocardial infarction (MI).MethodsThe TIDE (Thrombus and Inflammation in sudden DEath) study included patients with angiographically-proven acute coronary occlusion as the cause of a ST elevation MI (STEMI) complicated by Sudden Cardiac Death (SCD group) or not (STEMI group). Thrombi were obtained by thrombo-aspiration before primary percutaneous coronary stenting and analyzed with a quantitative method using scanning electron microscopy. We compared the composition of the thrombi responsible for the coronary occlusion between the two groups and evaluated factors influencing its composition.ResultsWe included 121 patients and found that thrombus composition was not different between the SCD group (n = 23) and the STEMI group (n = 98) regarding content of fibrin fibers (60.3 ± 18.4% vs. 62.4 ± 18.4% respectively, p = 0.68), platelets (16.3 ± 19.2% vs. 15.616.7 ±%, p = 0.76), erythrocytes (14.6 ± 12.5% vs. 13 ± 12.1%, p = 0.73) and leukocytes (0.6 ± 0.9% vs. 0.8 ± 1.5%, p = 0.93). Thrombus composition did not differ between patients receiving upstream-use of glycoprotein IIb/IIIa platelet receptor inhibitors (GPI) and patients free of GPI. The only factor found to influence thrombus composition was the ischemic time from symptom onset to primary PCI, with a decreased content in fibrin fibers (57.8 ± 18.5% vs. 71.9 ± 10.1%, p = 0.0008) and a higher platelet content (19.2 ± 19.1% vs. 7.9 ± 5.7% p = 0.014) in early presenters (6 h of ischemic time).ConclusionComposition of intracoronary thrombi in STEMI patients does not differ between those presenting with and without SCD. Time from symptom onset to coronary reperfusion seems to be the strongest factor influencing thrombus composition in M

The flexible C-terminal parts of fibrinogen's Aα chains named the αC regions have been shown to play a role in fibrin self-assembly, although many aspects of their structure and functions remain unknown. To examine the involvement of the αC regions in the early stages of fibrin formation, we used high-resolution atomic force microscopy to image fibrinogen and oligomeric fibrin. Plasma-purified full-length human fibrinogen or des-αC fibrinogen lacking most of the αC regions, untreated or treated with thrombin, was imaged. Up to 80% of the potentially existing αC regions were visualized and quantified; they were highly heterogeneous in their length and configurations. Conversion of fibrinogen to fibrin was accompanied by an increase in the incidence and length of the αC regions as well as transitions from more compact conformations, such as a globule on a string, to extended and more flexible offshoots. Concurrent dynamic turbidimetry, confocal microscopy, and scanning electron microscopy revealed that trimming of the αC regions slowed down fibrin formation, which correlated with longer protofibrils, thinner fibers, and a denser network. No structural distinctions, except for the incidence of the αC regions, were revealed in the laterally aggregated protofibrils made of the full-length or des-αC fibrinogens, suggesting a pure kinetic effect of the αC regions on the fibrin architecture. This work provides a structural molecular basis for the promoting role of the αC regions in the early stages of fibrin self-assembly and reveals this stage of fibrin formation as a potential therapeutic target to modulate the structure and mechanical properties of blood clots.

In-stent restenosis (ISR) and late stent thrombosis are the major complications associated with the use of metal stents and drug eluting stents respectively. Our lab previously investigated the use of peptide CD47 in improving biocompatibility of bare metal stents in a rat carotid stent model and our results demonstrated a significant reduction in platelet deposition and ISR. However, this study did not characterize the stability of the pepCD47 on metal surfaces post storage, sterilization and deployment. Thus, the objective of the present study was 1) to test the stability of the peptide post - storage, sterilization, exposure to shear and mechanical stress and 2) to begin to expand our current knowledge of pepCD47 coated metal surfaces into the preclinical large animal rabbit model. Our results show that the maximum immobilization density of pepCD47 on metal surfaces is approximately 350 ng/cm2. 100% of the pepCD47 was retained on the metal surface post 24 weeks of storage at 4 °C, exposure to physiological shear stress, and mechanical stress of stent expansion. The bioactivity of the pepCD47 was found to be intact post 24 weeks of storage and ethylene oxide sterilization. Finally our ex vivo studies demonstrated that compared to bare metal the rabbit pepCD47 coated surfaces showed - 45% reduced platelet adhesion, a 10-fold decrease in platelet activation, and 93% endothelial cell retention. Thus, our data suggests that pepCD47 coating on metal surfaces is stable and rabbit pepCD47 shows promising preliminary results in preventing thrombosis and not inhibiting the growth of endothelial cells. Statement of significance Biocompatibility of bare metal stents is a major challenge owing to the significantly high rates of in-stent restenosis. Previously we demonstrated that peptide CD47 functionalization improves the biocompatibility of bare metal stents in rat model. A similar trend was observed in our ex vivo studies where rabbit blood was perfused over the rabbit pepCD47 functionalized surfaces. These results provide valuable proof of concept data for future in vivo rabbit model studies. In addition, we investigated stability of the pepCD47 on metal surface and observed that pepCD47 coating is stable over time and resistant to industrially relevant pragmatic challenges.

The major structural component of a blood clot is a mesh of fibrin fibers. Our goal was to determine whether fibrinogen glycation and fibrin fiber diameter have an effect on the mechanical properties of single fibrin fibers. We used a combined atomic force microscopy/fluorescence microscopy technique to determine the mechanical properties of individual fibrin fibers formed from blood plasma. Blood samples were taken from uncontrolled diabetic patients as well as age-, gender-, and body-mass-index-matched healthy individuals. The patients then underwent treatment to control blood glucose levels before end blood samples were taken. The fibrinogen glycation of the diabetic patients was reduced from 8.8 to 5.0 mol glucose/mol fibrinogen, and the healthy individuals had a mean fibrinogen glycation of 4.0 mol glucose/mol fibrinogen. We found that fibrinogen glycation had no significant systematic effect on single-fiber modulus, extensibility, or stress relaxation times. However, we did find that the fiber modulus, Y, strongly decreases with increasing fiber diameter, D, as Y∝D−1.6. Thin fibers can be 100 times stiffer than thick fibers. This is unusual because the modulus is a material constant and should not depend on the sample dimensions (diameter) for homogeneous materials. Our finding, therefore, implies that fibrin fibers do not have a homogeneous cross section of uniformly connected protofibrils, as is commonly thought. Instead, the density of protofibril connections, ρPb, strongly decreases with increasing diameter, as ρPb∝D−1.6. Thin fibers are denser and/or have more strongly connected protofibrils than thick fibers. This implies that it is easier to dissolve clots that consist of fewer thick fibers than those that consist of many thin fibers, which is consistent with experimental and clinical observations.

The blood compatibility of various intravascular (IV) devices (e.g., catheters, sensors, etc.) is compromised by activation of platelets that can cause thrombus formation and device failure. Such devices also carry a high risk of microbial infection. Recently, nitric oxide (NO) releasing polymers/devices have been proposed to reduce these clinical problems. CD47, a ubiquitously expressed transmembrane protein with proven anti-inflammation/anti-platelet properties when immobilized on polymeric surfaces, is a good candidate to complement NO release in both effectiveness and longevity. In this work, we successfully appended CD47 peptides (pepCD47) to the surface of biomedical grade polyurethane (PU) copolymers. SIRPα binding and THP-1 cell attachment experiments strongly suggested that the pepCD47 retains its biological properties when bound to PU films. In spite of the potentially high reactivity of NO toward various amino acid residues in CD47, the efficacy of surface-immobilized pepCD47 to prevent inflammatory cell attachment was not inhibited after being subjected to a high flux of NO for three days, demonstrating excellent compatibility of the two species. We further constructed a CD47 surface immobilized silicone tubing filled with NO releasing S-nitrosoglutathione/ascorbic acid (GSNO/AA) solution for synergistic biocompatibility evaluation. Via an ex vivo Chandler loop model, we demonstrate for the first time that NO release and CD47 modification could function synergistically at the blood/material interface and produce greatly enhanced anti-inflammatory/anti-platelet effects. This concept should be readily implementable to create a new generation of thromboresistant/antimicrobial implantable devices.

Polyhedral erythrocytes, named polyhedrocytes, are formed in contracted blood clots and thrombi, as a result of compression by activated contractile platelets pulling on fibrin. This deformation was shown to be mechanical in nature and polyhedrocytes were characterized using light and electron microscopy. Through three-dimensional reconstruction, we quantified the geometry of biconcave, intermediate, and polyhedral erythrocytes within contracting blood clots. During compression, erythrocytes became less oblate and more prolate than the biconcave cells and largely corresponded to convex, irregular polyhedra with a total number of faces ranging from 10 to 16. Faces were polygons with 3 to 6 sides. The majority of the faces were quadrilaterals, though not all sides were straight and not all faces were flat. There were no changes in the surface area or volume. These results describe the gradual natural deformation of erythrocytes as a part of compaction into a tightly packed array that is an important but understudied component of mature blood clots and thrombi.

Essentials Factor VIII (FVIII) replacement in hemophilia A corrects bleeding due to low thrombin generation. There is uncertainty about the minimal or trough level of FVIII needed for optimal treatment. FVIII improves physical properties of hemophilia clots, including stiffness and permeability. Fibrinogen and blood cells also stabilize hemophilia clots to decrease bleeding risk. Background Factor VIII (FVIII) replacement is standard of care for patients with hemophilia A (HemA); however, patient response does not always correlate with FVIII levels. We hypothesize this may be in part due to the physical properties of clots and contributions of fibrin, platelets, and erythrocytes, which may be important for hemostasis. Objective To understand how FVIII contributes to effective hemostasis in terms of clot structure and mechanical properties Patients/Methods In vitro HemA clots in human plasma or whole blood were analyzed using turbidity waveform analysis, confocal microscopy, and rheometry with or without added FVIII. In vivo clots from saphenous vein puncture in wild-type and HemA mice with varying FVIII levels were examined using scanning electron microscopy. Results FVIII profoundly affected HemA clot structure and physical properties; added FVIII converted the open and porous fibrin meshwork and low stiffness of HemA clots to a highly branched and dense meshwork with higher stiffness. Platelets and erythrocytes incorporated into clots modulated clot properties. The clots formed in the mouse saphenous vein model contained variable amounts of compressed erythrocytes (polyhedrocytes), fibrin, and platelets depending on the levels of FVIII, correlating with bleeding times. FVIII effects on clot characteristics were dose-dependent and reached a maximum at ~25% FVIII, such that HemA clots formed with this level of FVIII resembled clots from unaffected controls. Conclusions Effective clot formation can be achieved in HemA by replacement therapy, which alters the architecture of the fibrin network and associated cells, thus increasing clot stiffness and decreasing clot permeability.

The present study extends our previous investigation of circulating antibody/fibrinogen/C1q complexes (FgIgC) associated with thrombosis in a heterophenotypic AαR16C proband, by focusing on the molecular and functional characteristics of the FgIgC, isolated by cryoprecipitation, FgIgC components were demonstrated by SDS-PAGE and by rotary shadowing electron microscopy. Affinity chromatography was used to isolate IgG and fibrinogen from FgIgC. Thrombin-induced clots were examined by scanning electron microscopy and turbidity measurements. IgG/fibrinogen binding was measured by ELISA. Fibrinogen Aα1-19 peptides, cleaved by thrombin from fragment N-DSK, were examined by mass spectrometry. Clot stiffness, platelet release of P-selectin, and fibrinogen self-assembly were assessed by thromboelastography, flow cytometry, and atomic force microscopy, respectively. The FgIgC effects included the following: increased P-selectin release from gel-sieved platelets, finer fiber networks and decreased stiffness of its clots, and marked inhibition of fibrinogen self-assembly. The abnormal proband fibrinogen structure displayed phosphorylated AαR16C-AαR16C homodimers and AαR16C-glutathione heterodimers. ELISA measurements disclosed pronounced binding by proband fibrinogen to proband IgG, which was blocked by the IgG's Fab fragment and by proband, but not by normal plasmic fragment E1. There was appreciable, but much weaker, binding to normal fibrinogen, to its fragments E1, and D1, and to homodimeric AαR16C fibrinogen. The antibody's primary target epitope included heterodimeric AαR16C-glutathione; a secondary epitope resided in the D region. Moreover, both the enhanced platelet activation (i.e. increased P-selectin release induced by FgIgC) and the highly phosphorylated FpA (i.e. resulting in its accelerated release by thrombin) may have contributed to the thrombotic diathesis.

SummaryInherited disorders of fibrinogen are rare and affect either the quantity (hypofibrinogenaemia and afibrinogenaemia) or the quality of the circulating fibrinogen (dysfibrinogenaemia) or both (hypodysfibrinogenaemia). Extensive allelic heterogeneity has been found for all these disorders: in congenital afibrinogenaemia for example more than 40 mutations, the majority in FGA, have been identified in homozygosity or in compound heterozygosity. Numerous mutations have also been identified in patients with hypofibrinogenaemia, many of these patients are in fact heterozygous carriers of afibrinogenaemia mutations. Despite the number of genetic analyses performed, the study of additional patients still allows the identification of novel mutations. Here we describe the characterization of a novel FGA intron 2 donor splice-site mutation (Fibrinogen Montpellier II) identified in three siblings with hypodysfibrinogenaemia. Functional analysis of RNA produced by the mutant minigene in COS-7 cells revealed that the mutation led to the in-frame skipping of exon 2. Western blot analysis of COS-7 cells expressing an exon 2 deleted FGA cDNA revealed that an alpha-chain lacking exon 2, which codes in particular for fibrinopeptide A and polymerisation knob ‘A’, has the potential to be assembled into a hexamer and secreted. Analysis of precipitated fibrinogen from patient plasma showed that the defect leads to the presence in the circulation of alpha-chains lacking knob ‘A’ which is essential for the early stages of fibrin polymerisation. Fibrin made from purified patient fibrinogen clotted with thrombin displayed thinner fibers with frequent ends and large pores.

Elevated levels of circulating fibrinogen are associated with an increased risk of atherothrombotic diseases although a causative correlation between high levels of fibrinogen and cardiovascular complications has not been established. We hypothesized that a potential mechanism for an increased prothrombotic state is the post-translational modification of fibrinogen by tyrosine nitration. Mass spectrometry identified tyrosine residues 292 and 422 at the carboxyl terminus of the β-chain as the principal sites of fibrinogen nitration in vivo. Immunoelectron microscopy confirmed the incorporation of nitrated fibrinogen molecules in fibrin fibers. The nitration of fibrinogen in vivo resulted in four distinct functional consequences: increased initial velocity of fibrin clot formation, altered fibrin clot architecture, increased fibrin clot stiffness, and reduced rate of clot lysis. The rate of fibrin clot formation and clot architecture was restored upon depletion of the tyrosine-nitrated fibrinogen molecules. An enhanced response to the knob “B” mimetic peptides Gly-His-Arg-Proam and Ala-His-Arg-Proam suggests that incorporation of nitrated fibrinogen molecules accelerates fibrin lateral aggregation. The data provide a novel biochemical risk factor that could explain epidemiological associations of oxidative stress and inflammation with thrombotic complications.

We detail for the first time the uniquely altered fibrin polymerization of homophenotypic Aalpha R16H dysfibrinogen. By polymerase chain reaction amplification and DNA sequencing, our new proposita's genotype consisted of a G>A transition encoding for Aalpha R16H, and an 11 kb Aalpha gene deletion. High-performance liquid chromatography disclosed fibrinopeptide A release approximately six times slower than its fibrinopeptide B. Turbidimetric analyses revealed unimpaired fibrin repolymerization, and abnormal thrombin-induced polymerization (1-7 mumol/l fibrinogen, > 96% coagulable), consisting of a prolonged lag time, slow rate, and abnormal clot turbidity maxima, all varying with thrombin concentration. For example, at 0.2-3 U/ml, the resulting turbidity maxima ranged from lower to higher than normal control values. By scanning electron microscopy, clots formed by 0.3 and 3 thrombin U/ml displayed mean fibril diameters 42 and 254% of the respective control values (n = 400). Virtually no such differences from control values were demonstrable, however, when clots formed in the presence of high ionic strength (micro = 0.30) or of monoclonal antibeta(15-42)IgG. The latter also prolonged the thrombin clotting time approximately three-fold. Additionally, thrombin-induced clots displayed decreased elastic moduli, with G' values of clots induced by 0.3, 0.7 and 3 thrombin U/ml corresponding to 11, 34, and 45% of control values. The results are consistent with increased des-BB fibrin monomer generation preceding and during polymerization. This limited the inherent gelation delay, decreased the clot stiffness, and enabled a progressively coarser, rather than finer, network induced by increasing thrombin concentrations. We hypothesize that during normal polymerization these constitutive des-BB fibrin monomer properties attenuate their des-AA fibrin counterparts.

Establishing the structure of the non-fibrillar collagens has provided a unique perspective to understanding their specialized functions in the extracellular matrix. These proteins exhibit very diverse conformations and supramolecular assemblies. Type XV collagen is a large macromolecule distinguished by a highly interrupted collagenous domain and many utilized sites of attachment for CS (chondroitin sulfate) and HS (heparan sulfate) glycosaminoglycan chains. It is present in most basement membrane zones of human tissues, where it is found closely associated with large collagen fibrils. To determine the molecular shape and organization of type XV, the protein was purified from human umbilical cords by salt extraction, and by ion-exchange and antibody-affinity chromatography. The representation of type XV in one of its most abundant tissue sources is estimated at only (1–2)×10−4% of dry weight. The molecules examined by transmission electron microscopy after rotary shadowing were visualized in multiple forms. Relatively few type XV monomers appeared elongated and kinked; most molecules were found in a knot/figure-of-eight/pretzel configuration not previously described for a collagen. Collective measurements of these populations revealed an average length of 193±16 nm. At the N-terminal end, identified by C-terminal antibody binding, were three 7.7 nm-diameter spheres, corresponding to TSPN-1 (N-terminal module of thrombospondin-1) modules, and attached to the collagen backbone by a short linker. The type XV monomers show the ability to self-assemble into higher-order structures. Some were arranged in complex clusters, but simpler oligomers, which may represent intermediates, were observed in a cruciform pattern with intermolecular binding sites that probably originate in the interruption sequences. The morphology of type XV is thus the antithesis of the fibrillar collagens, and the shape attains the required flexibility to form the spectrum of interconnecting links between banded fibrils at the basement membrane/interstitial border. These type XV structures may act as a biological ‘spring’ to stabilize and enhance resilience to compressive and expansive forces, and the multimers, in particular, with selective complements of many localized CS and HS chains, may be instrumental in spatial and temporal recruitment of modulators in growth, development and pathological processes.

Despite the importance of circulating microparticles in haemostasis and thrombosis, there is limited evidence for potential causative effects of naturally produced cell-derived microparticles on fibrin clot formation and its properties. We studied the significance of blood microparticles for fibrin formation, structure and susceptibility to fibrinolysis by removing them from platelet-free plasma using filtration. Clots made in platelet-free and microparticle-depleted plasma samples from the same healthy donors were analyzed in parallel. Microparticles accelerate fibrin polymerisation and support formation of more compact clots that resist internal and external fibrinolysis. These variations correlate with faster thrombin generation, suggesting thrombin-mediated kinetic effects of microparticles on fibrin formation, structure and properties. In addition, clots formed in the presence of microparticles, unlike clots from the microparticle-depleted plasma, contain 0.1–0.5-μm size granular and CD61-positive material on fibres, suggesting that platelet-derived microparticles attach to fibrin. Therefore, the blood of healthy individuals contains functional microparticles at the levels that have a procoagulant potential. They affect the structure and stability of fibrin clots indirectly through acceleration of thrombin generation and through direct physical incorporation into the fibrin network. Both mechanisms underlie a potential role of microparticles in haemostasis and thrombosis as modulators of fibrin formation, structure and resistance to fibrinolysis.

The effectiveness of endovascular stents is hindered by in-stent restenosis (ISR), a secondary re-obstruction of treated arteries due to unresolved inflammation and activation of smooth muscle cells in the arterial wall. We previously demonstrated that immobilized CD47, a ubiquitously expressed transmembrane protein with an established role in immune evasion, can confer biocompatibility when appended to polymeric surfaces. In present studies, we test the hypothesis that CD47 immobilized onto metallic surfaces of stents can effectively inhibit the inflammatory response thus mitigating ISR. Recombinant CD47 (recCD47) or a peptide sequence corresponding to the Ig domain of CD47 (pepCD47), were attached to the surfaces of both 316L-grade stainless steel foils and stents using bisphosphonate coordination chemistry and thiol-based conjugation reactions to assess the anti-inflammatory properties of CD47-functionalized surfaces. Initial in vitro and ex vivo analysis demonstrated that both recCD47 and pepCD47 significantly reduced inflammatory cell attachment to steel surfaces without impeding on endothelial cell retention and expansion. Using a rat carotid stent model, we showed that pepCD47-functionalized stents prevented fibrin and platelet thrombus deposition, inhibited inflammatory cell attachment, and reduced restenosis by 30%. It is concluded that CD47-modified stent surfaces mitigate platelet and inflammatory cell attachment, thereby disrupting ISR pathophysiology.

Incipient clot formation in whole blood and fibrin gels was studied by the rheometric techniques of controlled stress parallel superposition (CSPS) and small amplitude oscillatory shear (SAOS). The effects of unidirectional shear stress on incipient clot microstructure, formation kinetics and elasticity are reported in terms of the fractal dimension (df) of the fibrin network, the gel network formation time (TGP) and the shear elastic modulus, respectively. The results of this first haemorheological application of CSPS reveal the marked sensitivity of incipient clot microstructure to physiologically relevant levels of shear stress, these being an order of magnitude lower than have previously been studied by SAOS. CSPS tests revealed that exposure of forming clots to increasing levels of shear stress produces a corresponding elevation in df, consistent with the formation of tighter, more compact clot microstructures under unidirectional flow. A corresponding increase in shear elasticity was recorded. The scaling relationship established between shear elasticity and df for fibrin clots and whole blood confirms the fibrin network as the dominant microstructural component of the incipient clot in terms of its response to imposed stress. Supplementary studies of fibrin clot formation by rheometry and microscopy revealed the substantial additional network mass required to increase df and provide evidence to support the hypothesis that microstructural changes in blood clotted under unidirectional shear may be attributed to flow enhanced thrombin generation and activation. CSPS also identified a threshold value of unidirectional shear stress above which no incipient clot formation could be detected. CSPS was shown to be a valuable haemorheological tool for the study of the effects of physiological and pathological levels of shear on clot properties.

Unlike many other mammals spending a considerable amount of time in water, river otters (Lutra canadensis (Schreber, 1777)) do not have a thick layer of body fat. Instead, they have a very densely packed layer of thin underhairs. The structure of river otter hair was examined by scanning electron microscopy and polarizing light microscopy. Guard hairs were hollow and became thicker distally and then tapered to a point and had different cuticle scales in proximal and distal regions. The cuticle of the thin underhairs had a striking pattern of sharply sculpted fins with deep grooves between them; usually there were four fins at each level, rotated 45° with respect to those at an adjacent level. Underhairs varied in diameter and the scales were sometimes petal-shaped. Polarizing light microscopy images showed interlocking arrangements of the underhairs that help to impede the penetration of water. Also, these images showed that the grooves between fins or petals of underhairs entrap air bubbles. The structure of the hairs allows them to interact loosely with each other, despite variations in size and structure. Furthermore, the nature of the interactions between the fins and depressions allows space between the hairs that can trap air bubbles to increase the thermal insulation of the otter's coat.

Formation of nitric oxide-derived oxidants has been linked to development of atherosclerosis and associated thrombotic complications. Although systemic levels of protein nitrotyrosine predict risk for coronary artery disease, neither specific proteins targeted for modification nor functional consequences that might contribute to disease pathogenesis have been defined. Here we report a selective increase in circulating levels of nitrated fibrinogen in patients with coronary artery disease. Exposure of fibrinogen to nitrating oxidants, including those produced by the myeloperoxidase-hydrogen peroxide-nitrite system, significantly accelerates clot formation and factor XIII cross-linking, whereas exposure of fibrinogen to non-nitrating oxidants decelerates clot formation. Clots formed with fibrinogen exposed to nitrating oxidants are composed of large bundles made from twisted thin fibrin fibers with increased permeation and a decrease in storage modulus G' value, suggesting that these clots could be easily deformed by mechanical stresses. In contrast, clots formed with fibrinogen exposed to non-nitrating oxidants showed decreased permeation with normal architecture. Fibrinogen modified by exposure to physiologic nitration systems demonstrated no difference in the rate of plasmin-induced clot lysis, platelet aggregation, or binding. Thus, increased levels of fibrinogen nitration may lead to a pro-thrombotic state via acceleration in formation of fibrin clots. The present results may account, in part, for the association between nitrative stress and risk for coronary artery disease.

Objective— A splice variant of fibrinogen, γ′, has an altered C-terminal sequence in its gamma chain. This γA/γ′ fibrin is more resistant to lysis than γA/γA fibrin. Whether the physical properties of γ′ and γA fibrin may account for the difference in their fibrinolysis rate remains to be established. Methods and Results— Mechanical and morphological properties of cross-linked purified fibrin, including permeability (Ks, in cm 2 ) and clot stiffness (G′, in dyne/cm 2 ), were measured after clotting γA and γ′ fibrinogens (1 mg/mL). γ′/γ′ fibrin displayed a non-significant decrease in the density of fibrin fibers and slightly thicker fibers than γA/γA fibrin (12±2 fiber/10 −3 nm 3 versus 16±2 fiber/10 −3 nm 3 and 274±38 nm versus 257±41 nm for γ′/γ′ and γA/γA fibrin, respectively; P =NS). This resulted in a 20% increase of the permeability constant (6.9±1.7 10 −9 cm 2 versus 5.5±1.9 10 −9 cm 2 , respectively; P =NS). Unexpectedly, γ′ fibrin was found to be 3-times stiffer than γA fibrin (72.6±2.6 dyne/cm 2 versus 25.1±2.3 dyne/cm 2 ; P Conclusions— Fibrinolysis resistance that arises from the presence of γA/γ′ fibrinogen in the clot is related primarily to an increase of fibrin cross-linking with only slight modifications of the clot architecture.

Integrin αIIbβ3clusters on the platelet surface after binding adhesive proteins in a process that regulates signal transduction. However, the intermolecular forces driving integrin self-association are poorly understood. This work provides new insights into integrin clustering mechanisms by demonstrating how temperature and ligand binding interact to affect the oligomeric state of αIIbβ3. The ligand-free receptor, solubilized in thermostable octyl glucoside micelles, exhibited a cooperative transition at ∼43 °C, monitored by changes in intrinsic fluorescence and circular dichroism. Both signals changed in a direction opposite to that for global unfolding, and both were diminished upon binding the fibrinogen γ-chain ligand-mimetic peptide cHArGD. Free and bound receptors also exhibited differential sensitivity to temperature-enhanced oligomerization, as measured by dynamic light scattering, sedimentation velocity, and sedimentation equilibrium. Van't Hoff analyses of dimerization constants for αIIbβ3 complexed with cHArGD, cRGD, or eptifibatide yielded large, favorable entropy changes partly offset by unfavorable enthalpy changes. Transmission electron microscopy showed that ligand binding and 37 °C incubation enhanced assembly of integrin dimers and larger oligomers linked by tail-to-tail contacts. Interpretation of these images was aided by threading models for αIIbβ3 protomers and dimers based on the ectodomain structure of αvβ3. We propose that entropy-favorable nonpolar interactions drive ligand-induced integrin clustering and outside-in signaling.

SummaryA biophysical approach tested the hypothesis that tirofiban, like eptifibatide, perturbs GpIIb/IIIa structure. Tirofiban bound tightly to platelet GpIIb/IIIa (EC50 ∼ 24 nmol/L) and effectively inhibited platelet aggregation (IC50 ∼ 37 nmol/L) but blocked platelet adhesion to clotted fibrin only at much higher doses (IC50 ∼ 580 nmol/L). Electrophoretic analyses demonstrated that tirofiban protected GpIIb/IIIa from SDSinduced subunit dissociation. However, saturating tirofiban concentrations had little or no effect on GpIIb/IIIa secondary or tertiary structure, as determined by circular dichroic spectroscopy, dynamic light scattering, and sedimentation velocity measurements performed with purified receptors in octyl glucoside. Moderate dose-dependent effects on GpIIb/IIIa quaternary structure were detected by sedimentation equilibrium. Transmission electron microscopy showed minimal tirofiban-induced receptor activation or oligomerization. Thus, even at the increased concentrations needed to block platelet:fibrin adhesive interactions, tirofiban exhibited only limited effects on GpIIb/IIIa conformation and clustering. Our results provide new insights into the mechanisms and potential prothrombotic complications of integrin antagonists.

BACKGROUND Existing approaches for measuring hemostasis parameters require multiple platforms, can take hours to provide results, and generally require 1–25 mL of sample. We developed a diagnostic platform that allows comprehensive assessment of hemostatic parameters on a single instrument and provides results within 15 min using 0.04 mL of blood with minimal sample handling. METHODS T2 magnetic resonance (T2MR) was used to directly measure integrated reactions in whole blood samples by resolving multiple water relaxation times from distinct sample microenvironments. Clotting, clot contraction, and fibrinolysis stimulated by thrombin or tissue plasminogen activator, respectively, were measured. T2MR signals of clotting samples were compared with images produced by scanning electron microscopy and with standard reference methods for the following parameters: hematocrit, prothrombin time, clot strength, and platelet activity. RESULTS Application of T2MR methodology revealed conditions under which a unique T2MR signature appeared that corresponded with the formation of polyhedral erythrocytes, the dynamics and morphology of which are dependent on thrombin, fibrinogen, hematocrit, and platelet levels. We also showed that the T2MR platform can be used for precise and accurate measurements of hematocrit (%CV, 4.8%, R2 = 0.95), clotting time (%CV, 3.5%, R2 = 0.94), clot strength (R2 = 0.95), and platelet function (93% agreement with light transmission aggregometry). CONCLUSIONS This proof-of-concept study demonstrates that T2MR has the potential to provide rapid and sensitive identification of patients at risk for thrombosis or bleeding and to identify new biomarkers and therapeutic targets with a single, simple-to-employ analytic approach that may be suitable for routine use in both research and diverse clinical settings.

Contraction of blood clots is necessary for hemostasis and wound healing and to restore flow past obstructive thrombi, but little is known about the structure of contracted clots or the role of erythrocytes in contraction. We found that contracted blood clots develop a remarkable structure, with a meshwork of fibrin and platelet aggregates on the exterior of the clot and a close-packed, tessellated array of compressed polyhedral erythrocytes within. The same results were obtained after initiation of clotting with various activators and also with clots from reconstituted human blood and mouse blood. Such close-packed arrays of polyhedral erythrocytes, or polyhedrocytes, were also observed in human arterial thrombi taken from patients. The mechanical nature of this shape change was confirmed by polyhedrocyte formation from the forces of centrifugation of blood without clotting. Platelets (with their cytoskeletal motility proteins) and fibrin(ogen) (as the substrate bridging platelets for contraction) are required to generate the forces necessary to segregate platelets/fibrin from erythrocytes and to compress erythrocytes into a tightly packed array. These results demonstrate how contracted clots form an impermeable barrier important for hemostasis and wound healing and help explain how fibrinolysis is greatly retarded as clots contract.

The factors that contribute to pulmonary embolism (PE), a potentially fatal complication of deep vein thrombosis (DVT), remain poorly understood. Whereas fibrin clot structure and functional properties have been implicated in the pathology of venous thromboembolism and the risk for cardiovascular complications, their significance in PE remains uncertain. Therefore, we systematically compared and quantified clot formation and lysis time, plasminogen levels, viscoelastic properties, activated factor XIII cross-linking, and fibrin clot structure in isolated DVT and PE subjects. Clots made from plasma of PE subjects showed faster clot lysis times with no differences in lag time, rate of clot formation, or maximum absorbance of turbidity compared with DVT. Differences in lysis times were not due to alterations in plasminogen levels. Compared with DVT, clots derived from PE subjects showed accelerated establishment of viscoelastic properties, documented by a decrease in lag time and an increase in the rate of viscoelastic property formation. The rate and extent of fibrin cross-linking by activated factor XIII were similar between clots from DVT and PE subjects. Electron microscopy revealed that plasma fibrin clots from PE subjects exhibited lower fiber density compared with those from DVT subjects. These data suggest that clot structure and functional properties differ between DVT and PE subjects and provide insights into mechanisms that may regulate embolization.

Lipoprotein(a) is composed of low-density lipoprotein linked both covalently and noncovalently to apolipoprotein(a). The structure of lipoprotein(a) and the interactions between low-density lipoprotein and apolipoprotein(a) were investigated by electron microscopy and correlated with analytical ultracentrifugation. Electron microscopy of rotary-shadowed and unidirectionally shadowed lipoprotein(a) prepared without glycerol revealed that it is a nearly spherical particle with no large projections. After extraction of both lipoprotein(a) and low-density lipoprotein with glycerol prior to rotary shadowing, the protein components were observed to consist of a ring of density made up of nodules of different sizes, with apolipoprotein(a) and apolipoprotein B-100 closely associated with each other. However, when lipoprotein(a) was treated with a lysine analogue, 6-aminohexanoic acid, much of the apolipoprotein(a) separated from the apolipoprotein B-100. In 6-aminohexanoic acid-treated preparations without glycerol extraction, lipoprotein(a) particles had an irregular mass of density around the core. In contrast, lipoprotein(a) particles treated with 6-aminohexanoic acid in the presence of glycerol had a long tail, in which individual kringles could be distinguished, extending from the ring of apolipoprotein B-100. The length of the tail was dependent on the particular isoform of apolipoprotein(a). Dissociation of the noncovalent interactions between apolipoprotein(a) and low-density lipoprotein as a result of shear forces or changes in the microenvironment may contribute to selective retention of lipoprotein(a) in the vasculature.

The platelet integrin alphaIIbbeta3 is representative of a class of heterodimeric receptors that upon activation bind extracellular macromolecular ligands and form signaling clusters. This study examined how occupancy of alphaIIbbeta3's fibrinogen binding site affected the receptor's solution structure and stability. Eptifibatide, an integrin antagonist developed to treat cardiovascular disease, served as a high-affinity, monovalent model ligand with fibrinogen-like selectivity for alphaIIbbeta3. Eptifibatide binding promptly and reversibly perturbed the conformation of the alphaIIbbeta3 complex. Ligand-specific decreases in its diffusion and sedimentation coefficient were observed at near-stoichiometric eptifibatide concentrations, in contrast to the receptor-perturbing effects of RGD ligands that we previously observed only at a 70-fold molar excess. Eptifibatide promoted alphaIIbbeta3 dimerization 10-fold more effectively than less selective RGD ligands, as determined by sedimentation equilibrium. Eptifibatide-bound integrin receptors displayed an ectodomain separation and enhanced assembly of dimers and larger oligomers linked through their stalk regions, as seen by transmission electron microscopy. Ligation with eptifibatide protected alphaIIbbeta3 from SDS-induced subunit dissociation, an effect on electrophoretic mobility not seen with RGD ligands. Despite its distinct cleft, the open conformer resisted guanidine unfolding as effectively as the ligand-free integrin. Thus, we provide the first demonstration that binding a monovalent ligand to alphaIIbbeta3's extracellular fibrinogen-recognition site stabilizes the receptor's open conformation and enhances self-association through its distant transmembrane and/or cytoplasmic domains. By showing how eptifibatide and RGD peptides, ligands with distinct binding sites, each affects alphaIIbbeta3's conformation, our findings provide new mechanistic insights into ligand-linked integrin activation, clustering and signaling.

Factor XIII on activation by thrombin cross-links fibrin. A common polymorphism Val to Leu at position 34 in the FXIII A subunit is under investigation as a risk determinant of thrombosis. Because Val34Leu is close to the thrombin cleavage site, the hypothesis that it would alter the function of FXIII was tested. Analysis of FXIII subunit proteolysis by thrombin using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high-performance liquid chromatography showed that FXIII 34Leu was cleaved by thrombin more rapidly and by lower doses than 34Val. Mass spectrometry of isolated activation peptides confirmed the predicted single methyl group difference and demonstrated that the thrombin cleavage site is unaltered by Val34Leu. Kinetic analysis of activation peptide release demonstrated that the catalytic efficiency (kcat/Km) of thrombin was 0.5 for FXIII 34Leu and 0.2 (μmol/L)−1× sec−1 for 34Val. Presence of fibrin increased the catalytic efficiency to 4.8 and 2.2 (μmol/L)−1 × sec−1, respectively. Although the 34Leu peptide was released at a similar rate as fibrinopeptide A, the 34Val peptide was released more slowly than fibrinopeptide A but more quickly than fibrinopeptide B generation. Cross-linking of γ- and -chains appeared earlier when fibrin was incubated with FXIII 34Leu than with 34Val. Fully activated 34Leu and 34Val FXIII showed similar cross-linking activity. Analysis of fibrin clots prepared using plasma from FXIII 34Leu subjects by turbidity and permeability measurements showed reduced fiber mass/length ratio and porosity compared to 34Val. The structural differences were confirmed by electron microscopy. These results demonstrate that Val34Leu accelerates activation of FXIII by thrombin and consequently affects the structure of the cross-linked fibrin clot.

The differences between coarse and fine fibrin clots first reported by Ferry have been interpreted in terms of nonspecific ionic strength effects for nearly 50 years and have fostered the notion that fibrin polymerization is largely controlled by electrostatic forces. Here we report spectroscopic and electron microscopy studies carried out in the presence of different salts that demonstrate that this long-held interpretation needs to be modified. In fact, the differences are due entirely to the specific binding of Cl− to fibrin fibers and not to generic ionic strength or electrostatic effects. Binding of Cl− opposes the lateral aggregation of protofibrils and results in thinner fibers that are also more curved than those grown in the presence of inert anions such as F−. The effect of Cl− is pH dependent and increases at pH>8.0, whereas fibers grown in the presence of F− remain thick over the entire pH range from 6.5 to 9.0. From the pH dependence of the Cl− effect it is suggested that the anion exerts its role by increasing the pKa of a basic group ionizing around pH 9.2. The important role of Cl− in structuring the fibrin clot also clarifies the role played by the release of fibrinopeptide B, which leads to slightly thicker fibers in the presence of Cl− but actually reduces the size of the fibers in the presence of F−. This effect becomes more evident at high, close to physiological concentrations of fibrinogen. We conclude that Cl− is a basic physiological modulator of fibrin polymerization and acts to prevent the growth of thicker, stiffer, and straighter fibers by increasing the pKa of a basic group. This discovery opens new possibilities for the design of molecules that can specifically modify the clot structure by targeting the structural domains responsible for Cl− binding to fibrin.

The effect of fibrin matrix micromorphology on neurite growth was investigated by measuring the length of neurites growing in three-dimensional fibrin gels with well characterized micromorphologies. Dorsal root ganglia (DRGs) from 7-day chick embryos were entrapped and cultured in gels made from varying concentrations of fibrinogen (5-15 mg/mL) or calcium (2-10 mM). The length of growing neurites was measured with light videomicroscopy, and the number and diameter of fibrin fiber bundles were measured from scanning electron micrographs. An increase in fibrinogen concentration caused a decrease in the average fiber bundle thickness, an increase in the number of fiber bundles, and a marked decrease in neurite length. Gels made with different calcium concentrations had a similar range of variation in fibrin fiber bundle number or diameter, but these variations had little effect on neurite and associated nonneuronal cell outgrowth. These results provide insights into the process of neurite advance within fibrin and may be useful in the design of fibrin-based materials used for peripheral nerve regeneration. Furthermore, this study provides the first detailed experimental data on the micromorphology of fibrin matrices made from more than 5 mg/mL of fibrinogen and indicates that existing kinetic models of fibrin polymerization do not accurately predict fibrin structure at these higher concentrations.

Fibrinogen Caracas II is an abnormal fibrinogen involving the mutation of A alpha serine 434 to N-glycosylated asparagine. Some effects of this mutation on the ultrastructure of fibrinogen Caracas II molecules, fibers, and clots were investigated by electron microscopy. Electron microscopy of rotary shadowed individual molecules indicated that most of the alphaC domains of fibrinogen Caracas II do not interact with each other or with the central domain, in contrast to control fibrinogen. Negatively contrasted Caracas II fibers were thinner and less ordered than control fibers, and many free fiber ends were observed. Scanning electron microscopy of whole clots revealed the presence of large pores bounded by local fiber networks made up of thin fibers. Permeation experiments also indicated that the average pore diameter was larger than that of control clots. The viscoelastic properties of the Caracas II clot, as measured by a torsion pendulum, were similar to those of control clots. Both the normal stiffness and increased permeability of the Caracas II clots are consistent with the observation that subjects with this dysfibrinogenemia are asymptomatic.

Blood clot contraction or retraction has been implicated to play a significant role in hemostasis, reduction of thrombus volume, and wound healing. Clot contraction is driven by forces that are generated by platelets and transmitted by fibrin and results in volume shrinkage followed by the compaction of erythrocytes into the core of the blood clot, resulting in their mechanical deformation towards a polyhedral shape, giving rise to the term polyhedrocytes. Despite the fact that erythrocytes are a major component of blood clots, relatively little is known about the influence of the mechanical properties or deformability of erythrocytes on the process of clot contraction. Increased hematocrit reduces extent of clot contraction due to mechanical resilience of erythrocytes and it is likely that in addition to a volume fraction the stiffness of erythrocytes can also affect the extent and rate of clot contraction. Here we tested this assumption by using artificially or naturally stiffened erythrocytes that have pathophysiological implications. The reduced deformability of erythrocytes is associated with a number of pathological conditions, such as hypertension, diabetes mellitus, atherosclerosis and smoking, but perhaps one of the most well-known diseases associated with increased erythrocyte rigidity is sickle cell disease (SCD). Another example of naturally stiff erythrocyte membrane is that of llama or camel that have red blood cells with increased osmotic resistance. To assess the extent of clot contraction, we used an optical tracking methodology that allows for the quantitative tracking for clot size. To assess the influence of erythrocyte rigidity on clot contraction we also used scanning electron microscopy to evaluate deformations of the erythrocytes, including the presence of polyhedrocytes. Centrifugation of citrated blood can be used to mimic the contractile forces generated by platelets and has been shown to cause polyhedrocyte formation. Increasing the erythrocyte rigidity through their treatment with a low concentration of glutaraldehyde resulted in a decrease in polyhedrocyte formation and the requirement of larger centrifugal forces to observe erythrocyte deformation, suggesting that the mechanical properties of erythrocytes could influence the process of clot contraction. As residual glutaraldehyde may have unwanted effects on platelets, clot contraction experiments were completed using naturally stiffer erythrocytes from SCD patients and llama ovalocytes, which are stiffer than human erythrocytes due to the increased amount of the membrane cytoskeletal protein spectrin. SCD patients were only included in this study if they had Sickle Trait, SCD Hb SS, SCD Hb SC and have not received recent transfusions. The blood samples of SCD patients were examined and on average had a 53% decrease (p Collectively, these results demonstrate that erythrocyte mechanical properties can influence the process of clot contraction so that stiffer cells reduce the rate and extent of clot contraction. A better understanding of the role of erythrocyte deformability in the process of clot contraction has the potential to inform the development of more targeted treatments for limiting bleeding and thrombosis in patients who are prone to having altered erythrocyte content and mechanical properties of these highly abundant cells embedded into blood clots and thrombi. Disclosures Weisel: Bayer: Research Funding.

Fibrin polymerization is a necessary part of hemostasis but clots can obstruct blood vessels and cause heart attacks and strokes. The polymerization reactions are specific and controlled, involving strong knob-into-hole interactions to convert soluble fibrinogen into insoluble fibrin. It has long been assumed that clots and thrombi are stable structures until proteolytic digestion. On the contrary, using the technique of fluorescence recovery after photobleaching, we demonstrate here that there is turnover of fibrin in an uncrosslinked clot. A peptide representing the knobs involved in fibrin polymerization can compete for the holes and dissolve a preformed fibrin clot, or increase the fraction of soluble oligomers, with striking rearrangements in clot structure. These results imply that in vivo clots or thrombi are more dynamic structures than previously believed that may be remodeled as a result of local environmental conditions, may account for some embolization, and suggest a target for therapeutic intervention.