Design and reshaping of an scFv directed against human platelet glycoprotein VI with diagnostic potential

Blood platelets play a key role in physiological hemostasis and in thrombosis. As a consequence, platelet functional analysis is widely used in the diagnosis of hemorrhagic disorders as well as in the evaluation of thrombosis risks and of the efficacy of antithrombotics. Glycoprotein (GP) VI is a platelet-specific collagen-signaling receptor. Clinical studies suggest that increased GPVI expression is associated with a risk of arterial thrombosis. Conversely, GPVI deficiencies have been identified in patients with defective platelet responses to collagen. Currently, there is no standard test available for measuring GPVI expression, essentially because antibodies usually cross-link GPVI upon binding, leading to platelet activation and consecutive changes in GPVI expression. Here, we designed a recombinant monovalent antibody fragment (scFv) derived from an anti-GPVI monoclonal IgG, 3J24, with the characteristics required to analyze GPVI expression. Guided by in silico modeling and V-KAPPA chain analysis, a Protein L (PpL) recognition pattern was engineered in the scFv, making possible its purification and detection using PpL conjugates. The PpL affinity-purified scFv is functional. It retains GPVI-binding specificity and allows detection of platelet surface-expressed GPVI without inducing platelet activation. In conclusion, the reshaped scFv may be very useful in the development of diagnostic approaches.     

Co-activation of platelets by prolactin or leptin--pathophysiological findings and clinical implications.

Platelet activation is involved in the pathogenesis of atherosclerosis and venous thromboembolism, and might therefore be a possible link between the two entities. Prolactin and leptin have recently been recognized as potent co-activators of ADP-dependent platelet aggregation or P-selectin expression, and are therefore suspected as additional risk factors for both arterial and venous thrombosis. There are clinical situations that have a known association with higher prolactin or leptin levels (pregnancy, obesity or anti-psychotic therapy) and increased risk of thromboembolic events. We compared the impact of both hormones on platelet activation in vitro and in vivo, indicating that prolactin has a stronger effect on platelet activation as leptin in vitro and in vivo. We have also demonstrated that prolactin levels are increased in so called idiopathic thrombosis, and that conversely, patients with prolactinoma have an increased frequency of thrombosis during the hyperprolactinemic state, in a retrospective analysis. Moreover, we have demonstrated increased prolactin values in stroke and myocardial infarction. Prospective studies have yet to be performed to give this theory its final confirmation. The involvement of hormonal factors in platelet aggregation and venous or arterial thrombosis may have important clinical implications such as for risk stratification of patients with venous and arterial thrombosis or new therapeutic options such as decreasing pro-coagulant hormone levels in certain risk situations.     

Prevention of Postoperative Deep Venous Thrombosis and Pulmonary Embolism

Two series of patients observed clinically and by phlebography suggest that one agent known to produce a reduction in platelet aggregation in vitro might be useful in reducing the incidence of deep venous thrombosis and pulmonary embolism postoperatively. The agent used in this study was hydroxychlorquine sulphate.     

Development of a flow-through system to create occluding thrombus

Occlusive thrombosis accounts for many heart attacks and strokes. These acute events are difficult to catch in patients and animal test methods may be misleading because anti-thrombotic therapeutics often do not cross-react with different species. This paper presents a new flow-through system that leads to rapid occlusive thrombosis in arterial flow conditions. Whole porcine blood is perfused through a tubular test section. The growing thrombus is visualized in real time from early platelet attachment, through accumulation, to occlusion. The progression of flow rate reduction provides a clear distinguishing parameter between thrombus formation and embolization. Thrombus growth rate is a linear function of very high shear rate beyond 40,000 s(-1). The histology of the thrombus reveals predominantly platelet accumulation and growth as a rough surface with tendrils. This flow-through system may be useful for the economic testing of new anti-thrombosis therapies.     

Vascular Complications in Nephrotic Syndrome: Relationship to Steroid Therapy and Accelerated Thromboplastin Generation

Arterial thrombosis and renal vein thrombosis occurred in two men and one woman, respectively, treated with steroids for the nephrotic syndrome. Raised serum cholesterol occurred in one patient only. Though bleeding, clotting, and prothrombin times, as well as the platelet counts, were normal, the rate of thromboplastin generation was increased in all three patients. Adding heparin to the plasma of one patient slowed the rate, and suggested that the raised rate could be due to removal or suppression of such normal circulating coagulation inhibitors. The thromboplastin generation test seems to be useful in diagnosing and managing such hypercoagulable states, and may help in further investigations of their causes.     

Abnormalities of platelet aggregation in chronic myeloproliferative disorders

A large variety of platelet dysfunctions has been described in chronic myeloproliferative disorders. These abnormalities may be due to deficiency of platelet granules, arahidonic acid metabolism defects or platelet membrane glycoproteins abnormalities. In this study we intend to detect the incidence of platelet function defects in 76 patients with various types of chronic myeloproliferative disorders. The platelet activity was studied in vitro by measuring platelet aggregation in response to ADP, epinephrine, collagen, arachidonic acid and ristocetin. These results were subsequently correlated with bleeding time and clinical aspects (bleeding or thrombosis). We found complex changes in platelet response with all agonists, in varied proportions. These abnormalities include absent, decreased or abnormal platelet aggregation response. In a few cases we found a markedly decreased, almost absent platelet response to all agonists while in some patients a normal platelet aggregation was noted. The correlation between these results and template bleeding time, thrombotic or hemorrhagic events and the type of diseases was difficult to establish and sometimes conflictual. Despite this fact, we consider that investigating platelet aggregation may be useful not only for the assesment of the hemostatic balance in chronic myeloproliferative disorders but also for a better insight into cell abnormalities occuring in these pathologic conditions.     

Imaging thrombus with radiolabelled monoclonal antibody to platelets.

Indium-111-hydroxyquinoline labelled platelets, though useful in the detection of thrombus, have not gained widespread use owing to the time and technical skill required for their preparation. A study was therefore conducted evaluating a new method of imaging thrombus with platelets radiolabelled with a 111In labelled monoclonal antibody, P256, directed to the platelet surface glycoprotein complex IIb/IIIa. When the number of receptors occupied by P256 was less than 3% of the total available on the platelet surface platelet function, as assessed by platelet aggregometry, was undisturbed. P256 was radiolabelled with 111In using diethylenetriaminepenta-acetic acid, which achieved a specific activity of 185 MBq (5 mCi)/mg. No impairment of immunoreactivity was detected at this specific activity. Platelets were labelled with radiolabelled monoclonal antibody in vitro in two patients at a receptor occupancy of 6% and in vivo--that is, by direct intravenous injection of P256--in six patients at a receptor occupancy of 1%. In vivo recovery and biodistribution kinetics suggested that after in vitro labelling platelets were minimally activated. The 111In kinetics recorded after intravenous P256 suggested rapid and efficient radiolabelling of platelets and gave no indication of platelet activation. Of the six patients who received intravenous P256, three had documented thrombus, two of whom gave positive results on P256 platelet scintigraphy. The third subject had chronic deep venous thrombosis and was scintigraphically negative. Imaging thrombus using a radiolabelled monoclonal antibody directed to platelets appears to offer great potential as a simple, non-invasive approach to the diagnosis of thrombosis.     

Molecular mechanisms of prothrombotic risk due to genetic variations in platelet genes: Enhanced outside-in signaling through the Pro33 variant of integrin beta3

In recent years inherited variations in platelet proteins have emerged as potential risk factors that could predispose individuals to arterial thrombosis. Although many studies have examined the association of platelet gene polymorphisms with particular disease states, the underlying mechanisms by which most of these polymorphisms contribute to the pathophysiology of thrombosis have remained largely unexplored. This review will focus on the cellular and molecular features by which these genetic changes affect platelet physiology. Although many genes have been investigated in this regard, only the genes encoding integrins beta3 and alpha2, and the platelet Fc receptor, Fc(gamma)RIIA, have been studied in any depth. In some cases (such as integrin alpha2), evidence supports a quantitative trait locus. For other genes, nonsynonymous nucleotide substitutions lead to structural and functional consequences. A large portion of this review will focus on the widely studied Leu33Pro (Pl(A)) polymorphism of integrin beta3, and will consider the potential mechanisms by which the Pro33 polymorphism could induce a prothrombotic risk. A detailed understanding of how polymorphisms modulate platelet physiology will be important for understanding individual differences in response to antiplatelet therapy.     

Development of a platelet adhesion transport equation for a computational thrombosis model

Thrombosis is a significant issue for cardiovascular device development and use. While thrombosis models are available, very few are device-related and none have been thoroughly validated experimentally. Here, we introduce a surface adherent platelet transport equation into a continuum model to account for the biomaterial interface/blood interaction. Using a rotating disc system and polyurethane-urea material, we characterize steady and pulsatile flow fields using laser Doppler velocimetry. In vitro measurements of platelet adhesion are used in combination with the LDV data to provide further experimental validation. The rotating disc system is computationally studied using the device-induced thrombosis model with the surface platelet adherent transport equation. The results indicate that the flow field is in excellent agreement to the experimental LDV data and that the platelet adhesion simulations are in good agreement with the in vitro platelet data. These results provide good evidence that this transport equation can be used to express the relationship between blood and a biomaterial if the correct platelet adhesion characteristics are known for the biomaterial. Further validation is necessary with other materials.     

Genetic determinants of platelet function in thromboembolic diseases

Within the past decade our understanding of thromboembolic disorders has become even more sophisticated as recent discoveries have suggested the influence of gene variants on the development of atherosclerotic disease and arterial thrombosis. Candidate genes encode proteins involved in processes relevant to atherosclerosis, ranging from cholesterol metabolism to arterial thrombosis. Platelets are key elements in primary hemostasis, but also in arterial thrombosis. Moreover, a number of genetic polymorphisms of platelet proteins may also induce gain or loss of function, supporting a role predisposing some individuals to thrombotic events. However, after thousands of studies, much controversy remains whether individual platelet polymorphisms contribute to an increased likelihood of thromboembolic disorders. Although platelet polymorphisms are a promising addition to more established cardiovascular risk factors, identifying genetic variants as a single cause of cardiovascular disease would be an oversimplification; instead, the contribution of these polymorphisms should also be considered in the context of a multifactorial disease. Gene-gene and gene-environment studies would identify specific combinations associated with a high risk to suffer from these diseases. The platelet's genetic heterogeneity should also be considered in every aspect of clinical medicine, ranging from susceptibility to diseases, pathogenesis, and clinical outcome to diversity in responses to drug treatment (pharmacogenomics), and bleeding.     

门静脉高压症脾切断流术后血栓形成的临床研究

目的:探讨门静脉高压症脾切断流术后门静脉系统血栓形成的相关原因。方法:回顾性分析2010年4月-2011年12月我科450例因肝硬化门静脉高压症行脾切断流术患者的临床资料,应用超声多普勒检测手术前后门静脉血流速度、门静脉直径及脾静脉、肠系膜上静脉、门静脉血栓情况,用Logistic回归分析术前肝功能Child-Pugh分级、门静脉直径、门静脉血流速度、脾脏的质量及术后血小板数量与门静脉系统血栓形成的关系。结果:术前门静脉系统有血栓患者75例,占16.7%。术后门静脉血栓再形成率52.9%。Logistic单因素分析提示门静脉系统血栓形成与门静脉内径、门静脉血流速度、脾脏质量、血清总胆红素、术后血小板数量有关。多因素分析发现门静脉系统血栓形成与门静脉内径、门静脉血流速度、脾脏质量有关,而与血清总胆红素、术后血小板数量无关。结论:肝硬化门静脉高压症脾切除术后门静脉系统血栓形成与门静脉内径、门静脉血流速度、脾脏质量有关。     

Aspirin, Platelet Aggregation, and the Circadian Variation of Acute Thrombotic Events

The onset of several acute cardiovascular diseases occurs in a circadian pattern, with a peak incidence in the hours soon after awakening. This finding, coupled with laboratory data that confirm a surge in platelet activation during the early morning hours, suggests that acute changes in platelet aggregability may be an important trigger of thrombosis. Therefore, the efficacy of antiplatelet agents, such as aspirin, in reducing risks of vascular occlusion may result, at least in part, from a blunting of these short-term changes in platelet aggregability. In this review, clinical and laboratory evidence describing these cyclical changes is discussed, as is current evidence of the effects of aspirin on platelet function and the circadian variation of acute thrombosis.     

Aspirin,Platelet Aggregation,and the Circadian Variation of Acute Thrombotic Events

The onset of several acute cardiovascular diseases occurs in a circadian pattern, with a peak incidence in the hours soon after awakening. This finding, coupled with laboratory data that confirm a surge in platelet activation during the early morning hours, suggests that acute changes in platelet aggregability may be an important trigger of thrombosis. Therefore, the efficacy of antiplatelet agents, such as aspirin, in reducing risks of vascular occlusion may result, at least in part, from a blunting of these short-term changes in platelet aggregability. In this review, clinical and laboratory evidence describing these cyclical changes is discussed, as is current evidence of the effects of aspirin on platelet function and the circadian variation of acute thrombosis.     

Kindlin-3 is essential for integrin activation and platelet aggregation

Integrin-mediated platelet adhesion and aggregation are essential for sealing injured blood vessels and preventing blood loss, and excessive platelet aggregation can initiate arterial thrombosis, causing heart attacks and stroke. To ensure that platelets aggregate only at injury sites, integrins on circulating platelets exist in a low-affinity state and shift to a high-affinity state (in a process known as integrin activation or priming) after contacting a wounded vessel. The shift is mediated through binding of the cytoskeletal protein Talin to the beta subunit cytoplasmic tail. Here we show that platelets lacking the adhesion plaque protein Kindlin-3 cannot activate integrins despite normal Talin expression. As a direct consequence, Kindlin-3 deficiency results in severe bleeding and resistance to arterial thrombosis. Mechanistically, Kindlin-3 can directly bind to regions of beta-integrin tails distinct from those of Talin and trigger integrin activation. We have therefore identified Kindlin-3 as a novel and essential element for platelet integrin activation in hemostasis and thrombosis.     

Four-Dimensional Characterization of Thrombosis in a Live-Cell,Shear-Flow Assay: Development and Application to Xenotransplantation

Background

Porcine xenografts are a promising source of scarce transplantable organs, but stimulate intense thrombosis of human blood despite targeted genetic and pharmacologic interventions. Current experimental models do not enable study of the blood/endothelial interface to investigate adhesive interactions and thrombosis at the cellular level under physiologic conditions. The purpose of this study was to develop and validate a live-cell, shear-flow based thrombosis assay relevant to general thrombosis research, and demonstrate its potential in xenotransplantation applications.

Methodology/Principal Findings

Confluent wild-type (WT, n = 48) and Gal transferase knock-out (GalTKO, which resist hyperacute rejection; n = 11) porcine endothelia were cultured in microfluidic channels. To mimic microcirculatory flow, channels were perfused at 5 dynes/cm² and 37°C with human blood stained to fluorescently label platelets. Serial fluorescent imaging visualized percent surface area coverage (SA, for adhesion of labeled cells) and total fluorescence (a metric of clot volume). Aggregation was calculated by the fluorescence/SA ratio (FR). WT endothelia stimulated diffuse platelet adhesion (SA 65 ± 2%) and aggregation (FR 120 ± 1 a.u.), indicating high-grade thrombosis consistent with the rapid platelet activation and consumption seen in whole-organ lung xenotransplantation models. Experiments with antibody blockade of platelet aggregation, and perfusion of syngeneic and allo-incompatible endothelium was used to verify the biologic specificity and validity of the assay. Finally, with GalTKO endothelia thrombus volume decreased by 60%, due primarily to a 58% reduction in adhesion (P < 0.0001 each); importantly, aggregation was only marginally affected (11% reduction, P < 0.0001).

Conclusions/Significance

This novel, high-throughput assay enabled dynamic modeling of whole-blood thrombosis on intact endothelium under physiologic conditions, and allowed mechanistic characterization of endothelial and platelet interactions. Applied to xenogeneic thrombosis, it enables future studies regarding the effect of modifying the porcine genotype on sheer-stress-dependent events that characterize xenograft injury. This in-vitro platform is likely to prove broadly useful to study thrombosis and endothelial interactions under dynamic physiologic conditions.     

Scavenger receptors: Implications in atherothrombotic disorders

Scavenger receptors are modified lipoprotein binding receptors, expressed on the surface of a variety of cells including endothelial, macrophages and platelets. The most extensively studied class B scavenger receptors comprise of CD36 and SR-BI and have been found to bind to native and modified LDL. Interaction of modified LDL to CD36 accelerates foam cell formation, the key step in atherosclerotic plaque deposition. Recently scavenger receptors have also been implicated in thrombosis. Platelet CD36 serves as a sensor of oxidative stress and modulator of platelet reactivity under hyperlipidemic conditions thus, inducing prothrombotic signals. In contrast, targeting platelet SR-BI corresponds to reduce platelet hyperreactivity in hyperlipidemia suggesting that targeting these receptors could be a promising strategy for the treatment of atherothrombotic disorders.     

Endotoxin enhances microvascular thrombosis in mouse cremaster venules via a TLR4-dependent, neutrophil-independent mechanism

Endotoxemia promotes adhesive interactions between platelets and microvascular endothelium in vivo. We sought to determine whether endotoxin (lipopolysaccharide, LPS) modified platelet thrombus formation in mouse cremaster venules and whether Toll-like receptor 4 (TLR4) and neutrophils were involved in the response. Intravital videomicroscopy was performed in the cremaster microcirculation of pentobarbital-anesthetized mice; venular platelet thrombi were induced with a light/dye endothelial injury model. C57BL/6 mice treated with Escherichia coli endotoxin had enhanced rates of venular platelet thrombus formation: the time to microvessel occlusion was reduced by approximately 50% (P < 0.005) compared with saline-treated animals. Enhanced microvascular thrombosis was evident as early as 2 h after LPS administration. LPS had no effect on thrombosis in either of two mouse strains with altered TLR4 signaling (C57BL/10ScNJ or C3H/HeJ), whereas it enhanced thrombosis in the control strains (C57BL/10J and C3H/HeN). LPS also enhanced platelet adhesion to endothelium in the absence of light/dye injury. Platelet adhesion, but not enhanced thrombosis, was inhibited by depletion of circulating neutrophils. LPS failed to enhance platelet aggregation ex vivo and did not influence platelet P-selectin expression, a marker of platelet activation. These findings support the notion that endotoxemia promotes platelet thrombus formation independent of neutrophils and without enhancement of platelet aggregation, via a TLR4-dependent mechanism.     

Role of platelets in hemostasis and thrombosis.

Platelets are central to both normal hemostasis and abnormal thrombotic states along with the vessel wall, coagulation elements, and blood flow. The platelets play a pivotal role in the reaction that occurs after vessel injury, during which platelets first adhere to the vessel wall, undergo a release reaction and then aggregate, probably as a result of the materials released from platelets. These processes can be studied by a series of in vitro tests which form the basis of our knowledge of platelets in hemostasis. While the hemostatic plug is usually microscopic in size, this same plug (platelet thrombus) may contribute to the pathogenesis of several arterial diseases such as transient ischemic attacks, sudden blindness, sudden cardiac death and acute respiratory death syndrome. Careful microscopic examinations have shown that platelet aggregates may be found in the microcirculation which could affect vital structures such as the conduction system of the heart. Both anatomic and therapeutic evidence evidence suggests that platelets play a role in venous thrombosis. Recent evidence suggests increased levels of materials known to be released from platelets in patients with both arterial and venous thrombi along with increased platelet coagulant activities in patients with venous thrombosis.     

Involvement of bioantioxidants in thrombosis in mice

SUMMARY

An involvement of free radicals in thrombosis has been suggested previously. In order to further explore the role of free radicals and antioxidants in thrombosis, we have measured preventive (enzymes of the glutathione redox cycle) and chain-breaking antioxidants (vitamin E and C) in whole blood, platelets, neutrophils (PMNLs), heart and lung following collagen and adrenaline induced thrombosis in mice. A significant decrease in platelet glutathione (GSH) level (54%) and glutathione reductase activity was observed after thrombosis. In addition, GSH content in whole blood was also found to be reduced. In PMNLs, an increase in glutathione peroxidase activity and a four-fold elevation in vitamin C content was observed following thrombosis. However, levels of vitamin E and total thiol groups remained unchanged in both the cells and tissues. The results further suggest involvement of free radicals and PMNLs in thrombosis.     

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.