Effects of nitrite and far-red light on coagulation.

Nitric oxide, NO, has been explored as a therapeutic agent to treat thrombosis. In particular, NO has potential in treating mechanical device-associated thrombosis due to its ability to reduce platelet activation and due to the central role of platelet activation and adhesion in device thrombosis. Nitrite is a unique NO donor that reduces platelet activation in that it's activity requires the presence of red blood cells whereas NO activity of other NO donors is blunted by red blood cells. Interestingly, we have previously shown that red blood cell mediated inhibition of platelet activation by adenosine diphosophate (ADP) is dramatically enhanced by illumination with far-red light that is likely due to photolysis of red cell surface bound NO congeners. We now report the effects of nitrite, far-red light, and their combination on several measure of blood coagulation using a variety of agonists. We employed turbidity assays in platelet rich plasma, platelet activation using flow cytometry analysis of a fluorescently labeled antibody to the activated platelet fibrinogen binding site, multiplate impedance-based platelet aggregometry, and assessment of platelet adhesion to collagen coated flow-through microslides. In all cases, the combination of far-red light and nitrite treatment decreased measures of coagulation, but in some cases mono-treatment with nitrite or light alone had no effect. Perhaps most relevant to device thrombosis, we observed that platelet adhesions was inhibited by the combination of nitrite and light treatment while nitrite alone and far-red light alone trended to decrease adhesion, but the results were mixed. These results support the potential of combined far-red light and nitrite treatment for preventing thrombosis in extra-corporeal or shallow-tissue depth devices where the far-red light can penetrate. Such a combined treatment could be advantageous due to the localized treatment afforded by far-red light illumination with minimal systemic effects. Given the role of thrombosis in COVID 19, application to treatment of patients infected with SARS Cov-2 might also be considered.

Amotosalen/ultraviolet A pathogen inactivation technology reduces platelet activatability, induces apoptosis and accelerates clearance.

Amotosalen and ultraviolet A (UVA) photochemical-based pathogen reduction using the Intercept™ Blood System (IBS) is an effective and established technology for platelet and plasma components, which is adopted in more than 40 countries worldwide. Several reports point towards a reduced platelet function after Amotosalen/UVA exposure. The study herein was undertaken to identify the mechanisms responsible for the early impairment of platelet function by the IBS. Twenty-five platelet apheresis units were collected from healthy volunteers following standard procedures and split into 2 components, 1 untreated and the other treated with Amotosalen/UVA. Platelet impedance aggregation in response to collagen and thrombin was reduced by 80% and 60%, respectively, in IBS-treated units at day 1 of storage. Glycoprotein Ib (GpIb) levels were significantly lower in IBS samples and soluble glycocalicin correspondingly augmented; furthermore, GpIbα was significantly more desialylated as shown by Erythrina Cristagalli Lectin (ECL) binding. The pro-apoptotic Bak protein was significantly increased, as well as the MAPK p38 phosphorylation and caspase-3 cleavage. Stored IBS-treated platelets injected into immune-deficient nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice showed a faster clearance. We conclude that the IBS induces platelet p38 activation, GpIb shedding and platelet apoptosis through a caspase-dependent mechanism, thus reducing platelet function and survival. These mechanisms are of relevance in transfusion medicine, where the IBS increases patient safety at the expense of platelet function and survival.

In Vivo Imaging Reveals Significant Tumor Vascular Dysfunction and Increased Tumor Hypoxia-Inducible Factor-1α Expression Induced by High Single-Dose Irradiation in a Pancreatic Tumor Model.

PURPOSE: To investigate the effect of high-dose irradiation on pancreatic tumor vasculature and microenvironment using in vivo imaging techniques. METHODS AND MATERIALS: A BxPC3 pancreatic tumor xenograft was established in a dorsal skinfold window chamber model and a subcutaneous hind leg model. Tumors were irradiated with a single dose of 4, 12, or 24 Gy. The dorsal skinfold window chamber model was used to assess tumor response, vascular function and permeability, platelet and leukocyte adhesion to the vascular endothelium, and tumor hypoxia for up to 14 days after 24-Gy irradiation. The hind leg model was used to monitor tumor size, hypoxia, and vascularity for up to 65 days after 24-Gy irradiation. Tumors were assessed histologically to validate in vivo observations. RESULTS: In vivo fluorescence imaging revealed temporary vascular dysfunction in tumors irradiated with a single dose of 4 to 24 Gy, but most significantly with a single dose of 24 Gy. Vascular functional recovery was observed by 14 days after irradiation in a dose-dependent manner. Furthermore, irradiation with 24 Gy caused platelet and leukocyte adhesion to the vascular endothelium within hours to days after irradiation. Vascular permeability was significantly higher in irradiated tumors compared with nonirradiated controls 14 days after irradiation. This observation corresponded with increased expression of hypoxia-inducible factor-1α in irradiated tumors. In the hind leg model, irradiation with a single dose of 24 Gy led to tumor growth delay, followed by tumor regrowth. CONCLUSIONS: Irradiation of the BxPC3 tumors with a single dose of 24 Gy caused transient vascular dysfunction and increased expression of hypoxia-inducible factor-1α. Such biological changes may impact tumor response to high single-dose and hypofractionated irradiation, and further investigations are needed to better understand the clinical outcomes of stereotactic body radiation therapy.

Synchrotron microbeam irradiation induces neutrophil infiltration, thrombocyte attachment and selective vascular damage in vivo.

Our goal was the visualizing the vascular damage and acute inflammatory response to micro- and minibeam irradiation in vivo. Microbeam (MRT) and minibeam radiation therapies (MBRT) are tumor treatment approaches of potential clinical relevance, both consisting of parallel X-ray beams and allowing the delivery of thousands of Grays within tumors. We compared the effects of microbeams (25-100 µm wide) and minibeams (200-800 µm wide) on vasculature, inflammation and surrounding tissue changes during zebrafish caudal fin regeneration in vivo. Microbeam irradiation triggered an acute inflammatory response restricted to the regenerating tissue. Six hours post irradiation (6 hpi), it was infiltrated by neutrophils and fli1a(+) thrombocytes adhered to the cell wall locally in the beam path. The mature tissue was not affected by microbeam irradiation. In contrast, minibeam irradiation efficiently damaged the immature tissue at 6 hpi and damaged both the mature and immature tissue at 48 hpi. We demonstrate that vascular damage, inflammatory processes and cellular toxicity depend on the beam width and the stage of tissue maturation. Minibeam irradiation did not differentiate between mature and immature tissue. In contrast, all irradiation-induced effects of the microbeams were restricted to the rapidly growing immature tissue, indicating that microbeam irradiation could be a promising tumor treatment tool.

Síndrome de plaquetas pegajosas. Reporte de 2 casos en el servicio de Cirugía Oral y Maxilofacial Pediátrica de la Fundación Hospital de la Misericordia / Sticky platelet syndrome. A report of 2 cases

El síndrome de plaquetas pegajosas (SPP) es un trastorno que se traduce en una serie de fenómenos protrombóticos a expensas de hiperagregabilidad plaquetaria. Es un trastorno raro y, a pesar de que puede ser identificado eficazmente mediante agregometría plaquetaria, se desconoce la etiología. Algunas publicaciones indican que el 20% de los eventos vaso-oclusivos arteriales corresponden a casos asociados al SPP. El SPP constituye un trastorno inusual; los pocos reportes oscurecen la prevalencia de esta entidad. Tiene implicación en el fracaso de colgajos libres e injertos por la formación de microtrombos. El propósito de este artículo es presentar 2 casos remitidos al Servicio de Cirugía Oral y Maxilofacial Pediátrica. Adicionalmente, se presenta una revisión actualizada sobre la etiología y diagnóstico de esta condición (AU)

Sticky platelet syndrome (SPS) is a disorder that is associated with a series of arterial and venous prothrombotic events due to of platelet hyperaggregability. It's a rare condition, and even though there are efficient platelet aggregometry methods to diagnose the disorder, the underlying pathophysiology remains unclear. Some publications suggest that approximately 20% of vaso-occlusive events are of SPS origin. SPS is an unusual condition; the limited number of reports contributes to the lack of documentation regarding this syndrome. SPS is associated with maxillofacial free flap and graft failures due to thrombi formation. The purpose of this article is to present 2 cases of SPS seen in the Pediatric Oral and Maxillofacial Surgery Department together with a review on the etiology and diagnostic features of SPS (AU)

ß-radiation reduces the reactivity of extracellular matrix proteins in intravascular brachytherapy (IVBT), resulting in decreased platelet adhesion.

BACKGROUND: Intravascular Brachytherapy as a tool to reduce restenosis is thought to alter vascular wall biology and vessel wall protein function. Platelet accumulation is also indeed important in the genesis of restenosis. We examine the in vitro effects of beta-radiation on the certain vessel wall extra cellular matrix proteins. We hypothesized that vessel wall (proteins) had become less prone to thrombosis. METHODS: We examined platelet adhesion to 20-Gy beta radiation treated extra cellular matrix proteins under flow conditions. Platelet flow adhesion was evaluated or quantified by image analysis, aggregation size analysis using the Watershed program and real-time fluorescence images of thrombus formation. The effect of beta radiation on vWF was further showing by measuring the binding of domain-specific antibodies to radiation treated vWF. RESULTS: 20-Gy beta radiation significantly decreased platelet adhesion to extra cellular matrix protein; vWF and collagen Type III and had no effect on the adhesion upon fibrinogen and fibronectin. The beta-radiation affected mostly the AI, A2 and A3 domains of the vWF molecule on the surface, whereas the D'-D3 and B-C1 domains on the surface remain unaffected and suggesting a significant decrease in vWF binding capacity to the GPIb, heparin and collagen ligands. CONCLUSION: Beta radiation treatment can alter the reactivity of the certain vessel wall extra cellular matrix proteins, in particular vWF and collagen. The vessel wall may become less prone to platelet adhesion, which results in decrease thrombus formation. It might help to reduce the onset of acute coronary occlusion after the intervention.

Microenvironmental geometry guides platelet adhesion and spreading: a quantitative analysis at the single cell level.

To activate clot formation and maintain hemostasis, platelets adhere and spread onto sites of vascular injury. Although this process is well-characterized biochemically, how the physical and spatial cues in the microenvironment affect platelet adhesion and spreading remain unclear. In this study, we applied deep UV photolithography and protein micro/nanostamping to quantitatively investigate and characterize the spatial guidance of platelet spreading at the single cell level and with nanoscale resolution. Platelets adhered to and spread only onto micropatterned collagen or fibrinogen surfaces and followed the microenvironmental geometry with high fidelity and with single micron precision. Using micropatterned lines of different widths, we determined that platelets are able to conform to micropatterned stripes as thin as 0.6 µm and adopt a maximum aspect ratio of 19 on those protein patterns. Interestingly, platelets were also able to span and spread over non-patterned regions of up to 5 µm, a length consistent with that of maximally extended filopodia. This process appears to be mediated by platelet filopodia that are sensitive to spatial cues. Finally, we observed that microenvironmental geometry directly affects platelet biology, such as the spatial organization and distribution of the platelet actin cytoskeleton. Our data demonstrate that platelet spreading is a finely-tuned and spatially-guided process in which spatial cues directly influence the biological aspects of how clot formation is regulated.

Exposure of brain to high-dose, focused gamma rays irradiation produces increase in leukocytes-adhesion and pavementing in small intracerebral blood vessels.

OBJECTIVE: Radiosurgery is used to destroy a predetermined target within the brain, with minimal radiation injury to the surrounding tissue. We hereby present our in vivo model to study the effects of single-session, high-dose radiation on the cerebral vessels that are targeted with radiosurgery using the Leksell Gamma Knife. METHODS: The study was conducted in 29 adult male WT C57BL/6J mice weighing 21 to 28 g (6-8 wk old). The animals were exposed to 100 Gy single-session focused gamma ray irradiation using the Leksell Gamma Knife, and subsequently underwent intravital microscopy at different time intervals to study leukocytes and platelets adhesion patterns to the endothelium of the irradiated cerebral micro-vessels. RESULTS: The leukocyte adhesion response showed a bell-shaped curve upon quantitative analysis with a steady increase in the number of adherent cells during the first four hours and a subsequent plateau response that was maintained during the next 24 hours. The platelet adhesion response did not demonstrate any particular pattern similar to the leukocyte response. CONCLUSION: The experiment was able to establish in vivo increased leukocyte adhesion to the cerebral vascular endothelial cells in response to radiation injury and elaborate the time frame within which the leukocyte adhesion response increases, reaches a peak and then starts decreasing.

Effects of a new pathogen-reduction technology (Mirasol PRT) on functional aspects of platelet concentrates.

BACKGROUND: Several strategies are being developed to reduce the risk of pathogen transmission associated with platelet (PLT) transfusion. STUDY DESIGN AND METHODS: The impact of a new technology for pathogen reduction based on riboflavin plus illumination (Mirasol PRT, Navigant Biotechnologies, Inc.) at 6.2 and 12.3 J per mL on functional and biochemical characteristics of PLTs was evaluated. PLT concentrates (PCs) obtained by apheresis were treated with Mirasol PRT and stored at 22 degrees C. Modifications in major PLT glycoproteins (GPIbalpha, GPIV, and GPIIb-IIIa), adhesive ligands (von Willebrand factor [VWF], fibrinogen [Fg], and fibronectin), activation antigens (P-selectin and LIMP), and apoptotic markers (annexin V binding and factor [F]Va) were analyzed by flow cytometry. Adhesive and cohesive PLT functions were evaluated with well-established perfusion models. Studies were performed on the preparation day (Day 0) and during PCs storage (Days 3 and 5). RESULTS: Levels of glycoproteins remained stable during storage in PCs treated with 6.2 J per mL pathogen reduction technology (PRT) and similar to those observed in nontreated PCs. When 12.3 J per mL PRT was applied, however, levels of GPIbalpha moderately decreased on Days 3 and 5. VWF, Fg, and FVa were not modified in their expression levels, either by treatment or by storage period. Fibronectin appeared more elevated in all PRT samples. A progressive increase in P-selectin and LIMP expression and in annexin V binding was observed during storage of PRT-treated PCs. Functional studies indicated that 6.2 J per mL Mirasol PRT-treated PLTs preserved adhesive and cohesive functions to levels compatible with those observed in the respective control PCs. CONCLUSION: PLT function was well preserved in PCs treated with 6.2 J per mL Mirasol PRT and stored for 5 days.

Photon-modulated changes of cell attachments on poly(spiropyran-co-methyl methacrylate) membranes.

Spiropyran is a photoresponsive molecule, and nonionic spiropyran is reversibly changed by UV irradiation to a hydrophilic polar, zwitterionic merocyanine isomer, and back again by visible light irradiation. A copolymer of nitrobenzospiropyran and methyl methacrylate, poly(NSP-co-MMA) was used as a material with a photosensitive surface. UV irradiation of the photosensitive surface of poly(NSP-co-MMA)-coated glass plates decreased the water contact angles (11 +/- 1 degrees ) and increased diameter of a water drop relative to the unexposed surface. Light-induced detachment of platelets and mesenchymal stem (KUSA-A1) cells on poly(NSP-co-MMA)-coated glass plates was observed upon simple- and patterned-light irradiation, whereas no light-induced detachment of platelets and mesenchymal stem cells was observed on poly(methyl methacrylate)-coated glass plates. This is a result of the change from a closed nonpolar spiropyran to the polar zwitterionic merocyanine isomer induced by UV irradiation. Light-induced detachment of fibrinogen adsorbed on poly(NSP-co-MMA) coated glass plates was also observed in this investigation.

Irradiation increases the interactions of platelets with the endothelium in vivo: analysis by intravital microscopy.

Adhesion of platelets to the endothelium is believed to be a major factor contributing to thrombosis and vascular occlusion after radiotherapy or endovascular irradiation. In the present study, platelet-endothelium interactions were analyzed in vivo by intravital microscopy in mesenteric venules of mice according to three parameters: (1) platelet rolling, (2) platelet adhesion, and (3) the presence of platelet clusters. A 10-Gy total-body irradiation of mice resulted in an increase in the frequency of appearance of these three types of platelet-endothelium interactions in postcapillary venules 6 and 24 h after exposure, whereas only minor alterations were seen in large venules. In addition, the duration of platelet adhesion was increased 24 h after irradiation in both postcapillary and large venules. However, P-selectin was not up-regulated on the platelet membrane and platelet-leukocytes were not seen rolling together, suggesting that changes in platelet-endothelial cell interaction result from endothelial cell activation rather than platelet activation. Our data suggest that irradiation transforms resting endothelial cells to a pro-adhesive surface for platelets, which could ultimately lead to thrombosis.

Blood irradiation by He-Ne laser induces a decrease in platelet responses to physiological agonists and an increase in platelet cyclic GMP.

The effect of He-Ne laser irradiation on platelet adhesion, activation and aggregation was investigated. Citrated whole blood was irradiated in vitro by He-Ne laser (632.8 nm, 7 mW) and then subjected to shear stress (1300 s-1) on subendothelial extracellular matrix (ECM)-coated plates. Laser irradiation was followed by a decrease in platelet adhesion and aggregation on ECM under flow conditions in a time exposure-dependent manner (by 30-40%). The inhibiting effect of laser light on platelets was detectable up to 1 h after the termination of irradiation. Laser irradiation of either platelet-rich plasma, gel-filtered platelets, platelet-poor plasma, or packed blood cells followed by whole blood reconstitution revealed a marked decrease in platelet deposition on ECM only in the cases of platelet-rich plasma or gel filtered platelets. In conventional aggregometry, laser-treated platelet-rich plasma demonstrated a diminished platelet response to both thrombin receptor-activating peptide (TRAP), converting a two-wave aggregation curve to reversible, and to the protein kinase C activator PMA (by 45%). In flow cytometry analysis, irradiated platelets presented lower fibrinogen binding and P-selectin expression in response to TRAP. Laser irradiation had no additional inhibitory effect on dibutyryl cGMP- and dibutyryl cAMP-pretreated platelets. A 50% increase in cGMP level was observed in laser-treated gel filtered platelets, both in the presence and in absence of the phosphodiesterase inhibitor, isobuthylmethylxanthine. The results suggest that guanylate cyclase is one of the primary mediators of the laser effect on platelet function.

Ionizing radiation enhances platelet adhesion to the extracellular matrix of human endothelial cells by an increase in the release of von Willebrand factor.

The effect of radiation on the secretion of von Willebrand factor by endothelial cells was studied in a three-compartment culture system. The release of von Willebrand factor was significantly increased at 48 h after a single gamma-radiation dose of 20 Gy in both the luminal and abluminal direction by 23 (P < 0.05) and 41% (P < 0.02), respectively. To establish whether the enhanced production of von Willebrand factor affected the thrombogenicity of the extracellular matrix, platelet adhesion to the matrix produced by a monolayer of cultured endothelial cells during 48 h after irradiation was analyzed in a perfusion chamber at high shear rate (1300 s-1). Platelet adhesion was significantly increased by irradiation both in the presence and in the absence of plasmatic von Willebrand factor by 65 (P < 0.05) and 34.5% (P < 0.005), respectively. Incubation of the perfusate with a monoclonal antibody that blocks the binding of von Willebrand factor to platelet GPIb (CLB-RAg 35) resulted in an almost complete inhibition of platelet adhesion. These data indicate that radiation enhances platelet adhesion to the the extracellular matrix by an increase in the release of von Willebrand factor by endothelial cells. This event may be important in early radiation-induced vascular pathology.

Thermal laser arterial injury and prostacyclin administration in dogs: thrombotic and hyperplastic consequences.

Angioplasty is considered as an alternative to surgical reconstruction of arteriosclerotic vessels especially since lasers and atherectomy devices have become clinically available. However, the resulting arterial injury may lead to acute thrombotic occlusion and chronic restenosis because of hyperplastic vascular repair. The purpose of this experimental study was to evaluate the consequences of thermal laser arterial injury on platelet deposition and myointimal hyperplasia in dog femoral arteries. An intraarterial, short-term prostacyclin (PGI2) infusion was given to evaluate the antithrombotic and antiproliferative effects of this drug. Severe arterial necrosis, partly carbonized and vacuolized, extending to the adventitia was induced by a transluminal heated laser probe motion. The platelet deposition after one hour was 33.62 +/- 6.56 (x 10(6)/cm2.) (mean +/- SEM) without prostacyclin, after 40 ng/kg/min prostacyclin (PGI2) 24.70 +/- 5.45 and after 400 ng/kg/min 9.3 +/- 2.26 (p less than 0.005 no PGI2 vs 400 ng/kg/min PGI2). Myointimal hyperplasia was present eight weeks after thermal laser vascular injury independent of the initially administered prostacyclin. In conclusion, acutely thrombotic and chronically hyperplastic femoral arteries were found following transluminal thermal arterial injury in dogs. Prostacyclin administration could be clinically beneficial in reducing acute vascular thrombosis following thermal angioplasty. Short-term use of this substance, however, may not prevent a hyperplastic response to angioplasty.

[Comparison of the thrombogenic properties of vascular wall after exposure to different lasers]. / Sravnenie trombogennykh svoistv sosudistoi stenki posle deistviia razlichnykh lazerov.

Quantitative thrombogenic assessment of the inner arterial surface after laser exposure was made in 33 experiments with peripheral arterial strips of dogs. In one experiment, vascular strips with native, mechanically de-endotheliazed or laser-damaged surface were consecutively placed in a platelet-enriched plasma recirculation system, after which platelet adhesion was estimated on vascular surface of the three types, using a scanning electron microscope, and the relative thrombogenic index was calculated. The laser models used included: 1) the AIG-neodymium model, 1.06 micron; 2) the argon model with visible (488 and 514 nm) and ultraviolet (350 nm) spectra; 3) the XeCl excimer type, 308 nm, as well as a preheated metal rod, imitating a ceramic or metal tip of the light guide outlet. The thrombogenic index was 83 +/- 7; 72 +/- 8; 57 +/- 9; 63 +/- 7 and 82 +/- 9%, respectively, for the above types of exposure, the differences being virtually insignificant. It is suggested that, irrespective of the type of laser employed, laser angioplasty should at least be accompanied with intensive anticoagulant therapy.

Prostacyclin synthesis in irradiated endothelial cells cultured from bovine aorta.

Confluent monolayers of bovine aortic endothelial cells were examined 2-72 h after exposure to 0.5-5.0 Gy of 60Co gamma-rays. Accumulation of prostacyclin [PGI2, measured as 6-ketoprostaglandin F1 alpha (6-keto-PGF1 alpha)] in the culture media and PGI2 production stimulated by exogenous arachidonate were correlated with cell detachment and release of lactate dehydrogenase (LDH) activity. Platelet adherence to irradiated and control monolayers also was studied. There were simultaneous time- and dose-dependent increases in cell detachment and in the titers of 6-keto-PGF1 alpha and LDH activity in the culture medium. These changes were evident between 4 and 8 h after 5 Gy or at 24 h after 0.5 Gy. Four hours after 5 Gy, both adherent and detached endothelial cells showed a twofold increase in PGI2 production during a 15-min incubation with arachidonate (10 microM). However, by 72 h this increase was less significant. The accumulation of 6-keto-PGF1 alpha appeared to be related to cell destruction, but radiation also stimulated PGI2 synthesis independent of cell detachment. There was an increased platelet interaction with irradiated monolayers, as a result of platelet adherence to subendothelial matrix exposed after cell detachment. However, irradiation did not alter the nonadherent property of the endothelial cell surface toward platelets.

[Changes in various blood platelet function tests in rabbits exposed to whole-body irradiation]. / Veränderungen einiger Tests der Blutplättchenfunktion bei ganzkörperbestrahlten Kaninchen.

With a selected spectrum of coagulation tests the functioning capacity of thrombocytes was investigated in rabbits exposed to a whole body irradiation by means of 60Co radiation with a LD 5/30. A reduced retraction could be proved for times of irradiation (the 5th, 8th, 11th, 21st, 35th, and 56th day). A reduced formation of malondialdehyde could be identified in thrombocytes on the 8th and 21st day after irradiation. No changes could be found in determining adhesiveness, platelet aggregation caused by ADP, and PF3A and PF3F tests. In the course of additional investigations (coagulation time in unprepared and siliconized glass tubes, thromboelastogramme, activated partial chromboplastine time), significant changes of coagulation time could be observed in siliconized glass tubes on the 8th, 11th, 21st, and 56th day following irradiation.