Parallel-plate Perfusion Chamber Research Articles

Matrix metalloproteinase-2 enhances platelet deposition on collagen under flow conditions.

Platelets contain and release matrix metalloproteinase-2 (MMP-2) that in turn potentiates platelet aggregation. Platelet deposition on a damaged vascular wall is the first, crucial, step leading to thrombosis. Little is known about the effects of MMP-2 on platelet activation and adhesion under flow conditions. We studied the effect of MMP-2 on shear-dependent platelet activation using the O'Brien filtration system, and on platelet deposition using a parallel-plate perfusion chamber. Preincubation of human whole blood with active MMP-2 (50 ng/ml, i.e. 0.78 nM) shortened filter closure time (from 51.8 ± 3.6 sec to 40 ± 2.7 sec, p<0.05) and increased retained platelets (from 72.3 ± 2.3% to 81.1 ± 1.8%, p<0.05) in the O'Brien system, an effect prevented by a specific MMP-2 inhibitor. High shear stress induced the release of MMP-2 from platelets, while TIMP-2 levels were not significantly reduced, therefore, the MMP-2/TIMP-2 ratio increased significantly showing enhanced MMP-2 activity. Preincubation of whole blood with active MMP-2 (0.5 to 50 ng/ml, i.e 0.0078 to 0.78 nM) increased dose-dependently human platelet deposition on collagen under high shear-rate flow conditions (3000 sec⁻¹) (maximum +47.0 ± 11.9%, p<0.05, with 50 ng/ml), while pre-incubation with a MMP-2 inhibitor reduced platelet deposition. In real-time microscopy studies, increased deposition of platelets on collagen induced by MMP-2 started 85 sec from the beginning of perfusion, and was abolished by a GPIIb/IIIa antagonist, while MMP-2 had no effect on platelet deposition on fibrinogen or VWF. Confocal microscopy showed that MMP-2 enhances thrombus volume (+20.0 ± 3.0% vs control) rather than adhesion. In conclusion, we show that MMP-2 potentiates shear-induced platelet activation by enhancing thrombus formation.

Recombinant VWF Promotes Platelet Adhesion Under Shear Stress Dependent on Its Multimer Size

Baxter has developed BAX 111, a recombinant von Willebrand factor (rVWF) currently being evaluated for the treatment of von Willebrand disease based on the results of a phase 3 clinical trial. BAX 111 contains ultra-large molecular weight multimers (ULMWM), which are the most hemostatically active type of multimer. ULMWM demonstrate higher functional activity than the multimers present in commercially available plasma-derived VWF (pdVWF) products in terms of VWF:RCo and VWF:CB activity. To investigate the contribution of VWF multimer size on BAX 111 hemostatic activity, fractions containing distinct portions of VWF multimers were generated and analyzed for their ability to mediate platelet adhesion under shear stress in vitro.rVWF fractions of varying VWF multimer size were purified by size-exclusion chromatography; size distribution was determined using low and high resolution agarose gel electrophoresis. A parallel-plate perfusion chamber system was used to investigate the rVWF-mediated adhesion of platelets from human blood to fibrillar collagen type I under shear stress. For this purpose, a VWF sample of 1.0 IU/mL VWF:Ag was added to healthy donor blood and the time course of surface coverage was determined by microscopy using fluorescent labeled platelets. Multiple pictures were taken every 5 seconds. Perfusion was performed for 3 minutes at +37°C and at a defined wall shear rate of 1500 s-1. Different batches of BAX 111, rVWF fractions of distinct multimer size, and three commercially available pdVWF products with low and high FVIII content were investigated.The endogenous VWF present in human whole blood mediated initial platelet adhesion with approximately 5% surface coverage after 120 seconds of perfusion. Additionally spiking human blood with 1.0 IU/mL of BAX 111 or rVWF fractions of different multimeric size resulted in increased, time-dependent platelet binding. The amount of platelets that became attached to collagen was clearly dependent on the size of rVWF. While highly multimerized BAX 111 effectively promoted platelet binding, lower-sized rVWF fractions containing VWF multimers of lower molecular weight showed decreased platelet adhesion properties. pdVWF products lacking ultra-large molecular weight multimers were less effective in mediating platelet adhesion to collagen, as observed by delayed platelet binding kinetics. After 60 seconds of perfusion, approximately twice the surface coverage was measured for BAX 111 compared to pdVWF products. The amount of FVIII contained in the pdVWF products had no influence on platelet adhesion.These results demonstrate that the improved hemostatic activity of BAX 111 over pdVWF is due to the presence of ULMWM and confirms their importance in mediating intact platelet binding. DisclosuresSchrenk:Baxter Innovations GmbH, Vienna, Austria: Employment. Schreiner:Baxter Innovations GmbH, Vienna, Austria: Employment. Gritsch:Baxter Innovations GmbH, Vienna, Austria: Employment. Turecek:Baxter Innovations GmbH, Vienna, Austria: Employment. Dockal:Baxter Innovations GmbH, Vienna, Austria: Employment. Scheiflinger:Baxter Innovations GmbH, Vienna, Austria: Employment.

Contrast-enhanced molecular ultrasound differentiates endoglin genotypes in mouse embryos

Targeted ultrasound contrast imaging has the potential to become a reliable molecular imaging tool. A better understanding of the quantitative aspects of molecular ultrasound technology could facilitate the translation of this technique to the clinic for the purposes of assessing vascular pathology and detecting individual response to treatment. The objective of this study was to evaluate whether targeted ultrasound contrast-enhanced imaging can provide a quantitative measure of endogenous biomarkers. Endoglin, an endothelial biomarker involved in the processes of development, vascular homeostasis, and altered in diseases, including hereditary hemorrhagic telangiectasia type 1 and tumor angiogenesis, was the selected target. We used a parallel plate perfusion chamber in which endoglin-targeted (MBE), rat isotype IgG2 control and untargeted microbubbles were perfused across endoglin wild-type (Eng+/+), heterozygous (Eng+/-) and null (Eng-/-) embryonic mouse endothelial cells and their adhesion quantified. Microbubble binding was also assessed in late-gestation, isolated living transgenic Eng+/- and Eng+/+ embryos. Nonlinear contrast-specific ultrasound imaging performed at 21 MHz was used to collect contrast mean power ratios for all bubble types. Statistically significant differences in microbubble binding were found across genotypes for both in vitro (p<0.05) and embryonic studies (p<0.001); MBE binding was approximately twofold higher in Eng+/+ cells and embryos compared with their Eng+/- counterparts. These results suggest that molecular ultrasound is capable of reliably differentiating between molecular genotypes and relating receptor densities to quantifiable molecular ultrasound levels.

The impact of factor Xa inhibition on axial dependent arterial thrombus formation triggered by a tissue factor rich surface

This study was designed to assess the effect of Factor Xa antagonists on thrombus formation at various axial positions on a tissue factor rich surface under arterial blood flow conditions. Non-anticoagulated, flowing human blood, drawn directly from an antecubital vein, was perfused over a tissue factor coated cover slip in a parallel-plate perfusion chamber. Thrombus surface coverage, thrombus mean height and fibrin surface coverage were measured at six different axial positions by confocal microscopy. Both thrombus surface coverage and mean height decreased along the cover slip axis whereas the fibrin surface coverage increased. Pre-chamber treatment of blood with the direct Factor Xa inhibitors Razaxaban and 813893 resulted in significantly reduced thrombus and fibrin formation at all axial positions investigated (P<0.05). Thrombus and fibrin deposition in a laminar flow chamber changed with axial position with surface coverage measurements being more reproducible than thrombus mean height. Data were more reproducible towards the centre of the flow chamber than at the extremities. Razaxaban and 813893 inhibited thrombus and fibrin formation at the highest concentrations tested. No difference in drug effect was apparent at different axial positions. In conclusion, axial position influences the degree of thrombus and fibrin deposition with measurements being less reproducible at the extremities of the flow chamber. This technique may prove useful for analysing anti-thrombotic drug effects before progression to clinical trials.

Using Well-Plate Microfluidic Devices to Conduct Shear-Based Thrombosis Assays

Shear stress plays a critical role in regulating platelet adhesion and thrombus formation at the site of vascular injury. As such, platelets are often examined in vitro under controlled shear flow conditions for their hemostatic and thrombotic functions. Common shear-based platelet analyses include the evaluation of genetic mutants, inhibitory or experimental compounds, matrix substrates, and the effects of different physiological and pathological shear forces. There are several laboratory instruments widely used for studying shear flow, including cone and plate viscometers and parallel plate perfusion chambers. These technologies vary widely in the types of samples, substrates, blood volumes, and throughput that are involved. Here, we describe a microfluidic system for platelet analysis under shear flow. We used the devices to study thrombus formation on collagen I and von Willebrand factor. The system was also used to investigate dose response to the antiplatelet compound, Abciximab, under shear flow conditions with an emphasis on maximizing the number of data points per single patient sample. The presented method confers multiple advantages over conventional approaches. These include the ability to assess up to 24 conditions simultaneously in real time, maintain identical physical conditions across experiments, and use extremely low donor volumes.

Effect of Two Different Dialysis Membranes on Leukocyte Adhesion and Aggregation

Aims: We evaluated modifications in formation of heterotypic platelet-leukocyte aggregation induced by dialysis through cellulosic or synthetic membranes and evaluated the effects of such procedures promoting adhesive interactions between leukocytes and normal endothelial cells (ECs). Methods: Samples were obtained from arterial and venous lines at baseline, after 15 and 120 min of hemodialysis. Heterotypic aggregation was assessed using flow-cytometric techniques. Experiments to determine leukocyte adhesion to ECs were performed in parallel plate perfusion chambers at 450 s^–1. Results: Patients dialyzed with a cellulosic membrane showed a significantly higher baseline granulocyte heterotypic aggregation (median 22.5%, range 8.6–32%) versus healthy subjects (median 10%, range 3.2–14.6%; p < 0.05). Granulocyte heterotypic aggregation values remained increased throughout the hemodialysis session not only in the arterial line (median 18 and 24.5%, range 7–30 and 8.7–36% at 15 and 2 h, respectively) but also in the venous line (median 20 and 25%, range 8.6–32 and 11.5–35% at 15 min and 2 h, respectively). Basal lymphocytes heterotypic aggregation values observed in uremic patients were 6% (0.1–7.1%) versus 1.0% (0.5–2.8%) in the control group (p < 0.05). The increase remained during the hemodialysis session both in the arterial line (median 5 and 4%, range 0.2–14 and 0.5–7.1 % at 15 min and 2 h, respectively) and in the venous line (median 7 and 7%, range 1.4–14 and 0.5–10.6% at 15 min and 2 h). In contrast, patients dialyzed with a synthetic membrane showed a decreased basal granulocyte heterotypic aggregation compared to healthy subjects (median 3.5 vs. 10%, range 2.8–7 vs. 3.2–14.6%, respectively; p < 0.05). For lymphocytes, basal heterotypic aggregation values were 0.2% (range 0.1–0.5%) in dialyzed patients vs. 0.98% (range 0.5–2.8%) in healthy subjects (p < 0.05), without changes throughout the dialysis session. Changes in leukocyte adhesion during hemodialysis did not reach statistical significance with either hemodialysis membrane. Our studies confirm a differential activation of platelets and leukocytes depending on the nature of the dialysis membranes. However, activation of circulating cellular elements by hemodialysis procedures did not enhance cross-talk interactions between leukocytes and unaltered ECs.

Thrombus Formation On Apex of Arterial Stenoses: the Need for a Fluid High Shear Stenosis Diagnostic Device

This review is focused upon the studies of thrombus formation in human non-anticoagulated blood on an apex of an eccentric stenosis positioned in the blood flow channel of a parallel-plate perfusion chamber. Thrombus formation in blood from healthy individuals and patients with various bleeding disorders, as well as the effects of a diet supplement and pharmacological interventions, are discussed in view of thrombus-forming mechanisms under these complex blood-flow conditions. Hallmarks of this significantly enhanced thrombus formation are the apparent dependence on thrombin generation, shear-induced platelet activation, induction of platelet procoagulant activity and pronounced platelet microparticle formation that parallel the growth of these fibrin-rich thrombi. The development of miniature models of these blood-flow devices for diagnostic purposes is suggested for the assessment and monitoring of the efficacy of antithrombotic regimens in blood from patients with atherosclerotic disease in parallel with assessments of platelet microparticle formation, shear-induced platelet activation and platelet procoagulant activity.

Flow-based assays for global assessment of hemostasis. Part 2: current methods and considerations for the future.

KellSa s.a.s., Biella, Italy; Centre for Cardiovascular Sciences and MRC Centre for Immune Regulation, The Medical School, The University of Birmingham, Birmingham, UK; Department of Biomedical Engineering, University of Rochester, Rochester, USA; Department of Biochemistry, CARIM, University of Maastricht, Maastricht, The Netherlands; Dept of Physiology, McGill University, Montreal, Quebec,Canada/ affiliation with CYTORHEX Inc of Canada; Center for Molecular and Vascular Biology, University of Leuven, Leuven; Belgium.

Formation of Procoagulant Platelet Derived Microparticles through Platelet GPIb-Interaction with Von Willebrand Factor under High Shear Stress.

We describe here novel findings of the mechanism of initial platelet contact with immobilized von Willebrand factor (VWF) under high shear stress and how this leads to the formation of procoagulant platelet derived microparticles. In a parallel plate perfusion chamber whole blood was perfused over multimeric VWF or dimeric VWF A1 domain at shear rates between 2,000 s−1 and 40,000 s−1. Platelet attachment to VWF always occurred through glycoprotein Ibα receptors located in discrete adhesion points (DAPs), i.e. few limited membrane areas of 0.05 to 0.1 μm2 that arrested the platelets on the surface. The ongoing flow translocated such anchored platelets downstream, thus pulling membrane tethers from the intact and unstimulated platelet. Tethers could remain connected with the platelet body or be eventually severed, which occurred preferentially at shear rates above 6,000 s−1. Depending on the length of the severed membrane fragment they represented either isolated tethers or microparticles (arrowheads; see Figure below), the latter defined by a diameter of 50 to 100 nanometers. The shear rate threshold for microparticle formation was between 6,000 s−1 and 10,000 s−1. Immuno-fluorescence and immuno-electron microscopy showed glycoprotein Ibα clustered in DAPs of microparticles and tethers, i.e. the contact sites with the surface immobilized VWF. The microparticles also exhibited tissue factors on their surface and showed significant procoagulant activity measured by thrombin generation. We propose that after GPIbα anchoring to VWF in flowing blood passive mechanical pulling of membrane from platelets may generate platelet derived microparticles that can potentially support thrombogenesis. [Display omitted]

Preparation and Charaterization of MoAb SZ118 and SZ118-scFv Against Platelet Glycoprotein VI.

GlycoproteinVI (GPVI ), one of the platelet collagen receptors, plays an important role in platelet activation and thrombosis. GPVI participates in platelet adhesion to exposed collagen under high shear rate condition, and leads to platelet activation and thrombus formation. GPVI is widely recognized as a requisite factor for the formation of platelet aggregates on the collagen surface under blood flow. There were growing evidences showed that impairment of GPVI function could result in a long-term antithrombotic protection and less bleeding side-effect, which potentialized that GPVI might be an interesting target for safe anti-adhesion and anti-thrombotic therapy. Recently monoclonal antibodies directed against GPVI were developed, which impaired the interaction of platelets and collagen. A human soluble recombinant GPVI (rGPVI), consisting of extracellular domain of GPVI, was expressed in E. coli. After immunizing Balb/C mice with the purified rGPVI, a monoclonal antibody, SZ118, was developed. Meanwhile the gene encoding for the light-and heavy-chain variable regions of SZ118 had been cloned by RT-PCR from murine hybridoma cells. After ligated into the vector pET20b(+), expression plasmid pET20b(+)-SZ118-scFv was contructed, and SZ118-scFv fragment was expressed in E.coli. The parallel plate perfusion chamber and platelet aggregometer were used to study the effects of SZ118 and SZ118-scFv on the platelet adhesion and aggregation to collagen. The citrated whole blood with or without SZ118 was perfused over the surface of type I fibrillar collagen (Chrono Log, at 80μg/ml) at high shear rate (1000 s−1) for 5 min. The platelet aggregates were visualized by the phase-contrast microscope, the coverage rates of the platelet aggregates were measured. Monoclonal antibody SZ118 and SZ118-scFv could bind to human platelets exclusively by flow cytometry. SZ118 inhibited collagen-induced platelet aggregation in PRP in dose-dependent manner, nearly full inhibition being attained at the concentration of 110 ug•mL−1. Similarly SZ118-scFv inhibited collagen-induced platelet aggregation with maximum inhitition rate 84.3%±5.6%. And also SZ118 and SZ118-scFv had part effect on convulxin-induced platelet aggregation, but having little effect on ADP. After treating the citrated whole blood with SZ118 or SZ118-scFv at final concentration 50μg/ml at 37° for 10 min, the platelet adhesion on the surface of collagen was markedly inhibited with the coverage rates of 1.03%±0.22%, respectively (22.7%±5.7% for control), which indicated that the platelets adhesion to collagen was inhibited by 95.5%by SZ118 and 68.3% by SZ118-scFv. It was concluded that the MoAb SZ118 inhibited platelet aggregation to collagen and platelet adhesion to immobilized fibrillar collagen under high shear rate condition.

Superior efficacy of clopidogrel plus acetylsalicylic acid compared with extended-release dipyridamole plus acetylsalicylic acid in preventing arterial thrombogenesis in healthy volunteers

Introduction Recent ex vivo platelet aggregometry data indicate that clopidogrel 75 mg/day plus acetylsalicylic acid (ASA) 75 mg/day is a more potent antiplatelet regimen than the marketed combination of dipyridamole+ASA. The present study was designed to assess the antithrombotic effect of both dual antiplatelet regimens using a human ex vivo model of arterial thrombosis. Materials and methods This was a randomized, double-blind, placebo-controlled, crossover study. During two 10-day treatment periods separated by a 14-day washout period, 23 healthy male volunteers received once-daily clopidogrel 75 mg plus acetylsalicylic acid 75 mg, or twice-daily extended-release dipyridamole 200 mg plus acetylsalicylic acid 25 mg. Assessments were made at baseline and on Day 10 of each period. Arterial thrombus formation was induced ex vivo by exposing a collagen-coated surface in a parallel-plate perfusion chamber to native blood for 3 min (arterial wall shear rate 2600 s −1). Total platelet and fibrin deposition was determined by immunoenzymatic methods. Results Compared with baseline values, the mean inhibition of total platelet deposition was 63.9±5.9% with clopidogrel plus acetylsalicylic acid, compared with 18.4±5.6% for extended-release dipyridamole plus acetylsalicylic acid (67% reduction; 95% CI, 49–79%; p<0.0001). Corresponding figures for fibrin deposition were 64.9±4.8% and 18.3±9.7%, respectively (58% reduction; 95% CI, 45–67%; p<0.0001). Both treatments were well tolerated. Conclusions Clopidogrel plus acetylsalicylic acid showed significantly superior antithrombotic efficacy compared with extended-release dipyridamole plus acetylsalicylic acid in preventing arterial thrombogenesis in humans.

Improved hemocompatibility of poly(ethylene terephthalate) modified with various thiol-containing groups

Thiol groups were attached to polyethylene terephthalate (PET) to promote the transfer of a known platelet inhibitor, nitric oxide (NO), from nitrosated thiols naturally found in the body to PET, followed by the release of NO from PET to prevent platelet adhesion. In order to immobilize the most thiols on the modified polymer, the processing parameters used to attach the following three thiol containing groups were assessed: l-cysteine, 2-iminothiolane, and a cysteine polypeptide. When comparing the immobilized concentrations of thiol groups from each of the optimized processes the amount of immobilized thiol groups increased in order with the following groups: cysteine polypeptide <2-iminothiolane < l-cysteine. The effect of each optimized polymer on platelet adhesion was studied by in vitro experiments utilizing a parallel plate perfusion chamber. Platelets in the following solutions were tested: Tyrode's buffer, 7 μ m nitrosated bovine serum albumin in Tyrode's buffer, 50% plasma in Tyrode's buffer, and 50% whole blood in Tyrode's buffer. All of the polymers demonstrated a significant decrease in platelet adhesion compared to controls when exposed to the BSANO, plasma and whole blood solutions. The most significant decrease was for the l-cysteine modified polymer in the plasma solution with a 65% decrease.

Strenuous but not moderate exercise increases the thrombotic tendency in healthy sedentary male volunteers.

We have investigated the effect of moderate and strenuous exercise on experimental arterial thrombus formation in men. Thrombogenesis was measured in 15 sedentary healthy male volunteers at rest or immediately after two standardized exercise tests performed for 30 min on a bicycle ergometer. The exercises were performed at a constant load corresponding to either 50 or 70% maximal oxygen uptake. Thrombus formation was induced ex vivo by exposing a collagen-coated coverslip in a parallel plate perfusion chamber to native nonanticoagulated blood for 3 min. The shear rate at the collagen surface was 2,600 s(-1). Platelet and fibrin deposition was quantified by immunoenzymatic methods. The results show that moderate exercise did not affect arterial thrombus formation. In contrast, platelet thrombus formation on collagen was increased on the average by 20% after 30 min at 70% maximal oxygen uptake (P = 0.03). Fibrin deposition on collagen remained unchanged with exercise, regardless of its intensity. Thus, with the use of a clinically relevant human experimental model of thrombosis, the present study suggests that exercise of heavy intensity may increase the risk for arterial thrombogenesis in sedentary young healthy male volunteers.

Shear-dependent morphology of von Willebrand factor bound to immobilized collagen

AbstractWe have developed an immunogold von Willebrand factor (VWF) detection method that permits almost complete coverage of individual VWF molecules, and by this unequivocal localization and morphologic analysis of collagen-bound VWF by atomic force microscopy (AFM). Perfusion of gel filtration–purified VWF in parallel plate perfusion chambers over glass coverslips coated with calf skin collagen, followed by AFM imaging in air, enabled us to assess possible morphologic differences between VWF bound at low (0.07 N/m2 = 0.7 dynes/cm2) and high (4.55 N/m2 = 45.5 dynes/cm2) shear stresses. No significant differences in VWF morphology were found, the molecules were oriented almost randomly, and there were no clear signs of VWF “uncoiling” either at a high or at a low shear regime. After perfusing 1 µg/mL VWF for 5 minutes, surface coverage at high shear was almost twice the one seen at low shear, and some larger and more irregularly shaped VWF molecules could be seen at high shear. This difference disappeared, however, at 15 minutes of perfusion and was probably caused by diffusion kinetics. Moreover, the presence of 68 × 109/L washed fixed platelets in the perfusate did not have any visible effect on VWF morphology at high versus low shear stress. These findings suggest that shear stress does not influence significantly the overall molecular morphology of VWF during its binding to collagen-coated surface and are consistent with a constitutively expressed affinity of collagen-bound VWF for glycoprotein Ib.

Role of 4 platelet membrane glycoprotein polymorphisms on experimental arterial thrombus formation in men

AbstractThis study investigates whether the polymorphisms of 3 important platelet receptors affected experimental thrombus formation in men. Forty healthy male volunteers randomly recruited were genotyped for the variable number of tandem repeat (VNTR) of GPIbα, the −5T/C polymorphism in the Kozak sequence of GPIbα, the 807C/T polymorphism of GPIa, and the PlA1/PlA2polymorphism of GPIIb/IIIa. Platelet thrombus formation was induced ex vivo by exposing a collagen-coated coverslip in a parallel plate perfusion chamber to native blood for 4 minutes. The shear rates at the collagen surface were 650 and 2600 s−1. At 2600 s−1 platelet thrombus formation was significantly related only to the 807C/T polymorphism. In contrast, at 650 s−1thrombus formation was significantly altered only by the Kozak sequence polymorphism. The VNTR and the PlA1/PlA2polymorphisms did not influence thrombus formation. Thus, platelet thrombus formation is significantly influenced by genetic variations of the GPIbα and GPIa receptors. The effect of these polymorphisms was dependent on the blood flow rate.

Uremic Medium Disturbs the Hemostatic Balance of Cultured Human Endothelial Cells

We have investigated the ability of serum from uremic patients to modify the thrombogenic properties of the endothelium. The effects of uremic medium on the morphology of endothelial cells (ECs), and their resistance to flow was analyzed. The influence of uremic media on the reactivity of the extracellular matrix (ECM) generated by ECs towards normal platelets was evaluated in a parallel-plate perfusion chamber. Exposure of ECs to uremic medium resulted in abnormal cell morphology and signs of an accelerated growth. Detachment of ECs exposed to circulating blood was increased when cells had been grown with media supplemented with uremic serum (21% vs. 14% non exposed). Platelet deposition was significantly elevated on ECMs generated in the presence of uremic media (uremicECMs) (p<0.01 vs. control studies). Effects of uremic serum were not observed at short incubation periods (5 h) but were evident after 24 or 72 h of incubation. Northern blot analysis revealed increased expression of tissue factor (TF) mRNA in ECs exposed to uremic conditions. Immunocytochemical methods detected an augmented expression of TF antigen on uremic ECMs. Incubation of ECMs with an antibody to human tissue factor prevented the increase in platelet deposition observed in uremic ECMs, suggesting that the presence of TF in ECM could be responsible for the enhanced platelet deposition. Results from our study indicate that uremic medium impairs the antithrombotic functions of cultured endothelial cells.

The Effect of Platelet PlA Polymorphism on Experimental Thrombus Formation in Man Depends on Blood Flow and Thrombogenic Substrate

A number of studies have reported conflicting data on the association of the PlA1/PlA2 polymorphism of the GPIIIa gene and coronary syndromes. We have investigated the effect of this polymorphism on experimental platelet thrombus formation in man. Forty healthy male volunteers were genotyped for the PlA1/PlA2 polymorphism. Thrombus formation was induced ex vivo by exposing a tissue factor (TF) or a collagen-coated coverslip in a parallel plate perfusion chamber to native blood for 2 and 4 min. The shear rates at these surfaces were 650 and 2,600 s(-1). Platelet and fibrin deposition was quantified by immunoenzymatic methods. The frequencies of PlA1/PlA1 and PlA1/PlA2 genotypes were 52.5% and 47.5%, respectively. Ex vivo deposition of fibrin on TF was not affected by the PlA1/PlA2 polymorphism. However, the ex vivo platelet deposition at 650 s(-1) was higher in blood from PlA1/PlA1 individuals than in PlA1/PlA2 individuals (P= 0.008 at 4 min). On collagen, neither fibrin nor platelet deposition was significantly affected by the PlA1/PlA2 polymorphism. Platelet thrombus formation is significantly influenced by genetic variations in the GPIIIa platelet receptor. This effect depends on the blood flow properties and the nature of the thrombogenic stimulus.

Shear Stress Affects the Kinetics of Staphylococcus aureus Adhesion to Collagen

Staphylococcus aureus is a major human pathogen that has been shown to bind collagen under static conditions. However, many staphylococcal infections are hematogenously acquired and adhesion events may be influenced by shear stress. In this study, we used a dynamic experimental system consisting of a parallel-plate perfusion chamber and phase-contrast video microscope to study the effects of shear stress on the adhesion kinetics of intact S. aureus to collagen surfaces in vitro. The adhesion of S. aureus Phillips to collagen types I, II, and IV was investigated over a physiologically relevant range of wall shear stresses at 37 degrees C. S. aureus PH100, a collagen adhesin-deficient mutant strain, was used as a control strain for the experiments. We found that S. aureus Phillips could adhere to collagens I, II, and IV at wall shear stresses less than 15 dyn/cm(2) and that the kinetics of the adhesion process were wall shear stress-dependent. Similar studies with PH100 demonstrated that these cells are unable to adhere firmly to collagen surfaces. Transient interactions between PH100 and the collagen surfaces were observed at low levels of shear stress suggesting that S. aureus may also interact with collagen by an alternative mechanism that does not lead to firm adhesion.

Adherence of platelets under flow conditions results in specific phosphorylation of proteins at tyrosine residues.

Collagen is a powerful platelet activating agent that promotes adhesion and aggregation of platelets. To differentiate the signals generated in these processes we have analyzed the tyrosine phosphorylation occurring in platelets after activation with collagen in suspension or under flow conditions. For the suspension studies, washed platelets were activated with different concentrations of purified type I collagen (ColI). Studies under flow conditions were performed using two different adhesive substrata: ColI and endothelial cells extracellular matrix (ECM). Coverslips coated with ColI or ECM were perfused through a parallel-plate perfusion chamber at 800 s(-1) for 5 min. After activation of platelets either in suspension or by adhesion, samples were solubilized and proteins were resolved by electrophoresis. Tyrosine-phosphorylated proteins were detected in immunoblots by specific antibodies. Activation of platelet suspensions with collagen induced tyrosine phosphorylation before aggregation could be detected. Profiles showing tyrosine-phosphorylated proteins from platelets adhered on ColI or on ECM were almost identical and lacked proteins p95, p80, p66, and p64, which were present in profiles from platelets activated in suspension. The intensity of phosphorylation was quantitatively weaker in those profiles from platelets adhered on ECM. Results from the present work indicate that activation of platelets in suspension or by adhesion induces differential tyrosine phosphorylation patterns. Phosphorylation of proteins p90 and p76 may be related to early activation events occurring during initial contact and spreading of platelets. Considering that adhesion is the first step of platelet activation, studies on signal transduction mechanisms under flow conditions may provide new insights to understand the signaling processes taking place at earliest stages of platelet activation.

Protamine Sulphate Inhibits Platelet Membrane Glycoprotein Ib-von Willebrand Factor Activity

Platelet adhesion to the injured vessel wall is essential in haemostasis and thrombosis. This process involves the interaction of the platelet glycoprotein Ib (GPIb) with surface bound von Willebrand factor (vWF). Since synthetic polycationic peptides of the general formula (Arg)n, (Lys)n or (Arg-Lys)n inhibit GPIb-vWF interaction, they were suggested as potential antithrombotics. Protamine sulphate is a highly cationic polypeptide, arginine accounting for approximately 60% of the primary sequence, utilized to neutralize the anticoagulant effect of heparin after cardiac surgery. We have investigated potential effects of protamine sulphate on the function of GPIb-vWF. Addition of protamine sulphate to platelet-rich plasma (PRP), reduced significantly the GPIb-vWF activity as assessed by ristocetin-induced platelet agglutination. When protamine sulphate was added to PRP containing heparin, even at clinically relevant neutralizing doses the GPIb-vWF activity was reduced by 20-30% (p < 0.001). Protamine sulphate in excess of heparin nearly abolished the activity. Furthermore, the direct effect of protamine sulphate on collagen-induced platelet thrombus formation in non-anticoagulated human blood was investigated by employing an ex-vivo parallel-plate perfusion chamber device. Protamine sulphate (200 microg/mL) reduced platelet-collagen adhesion at shear rates of 650 and 2600 sec(-1) by 40% (p< 0.004) and 45% (p < 0.0001), respectively. The corresponding platelet thrombus volumes were concomitantly reduced by 90% (p < 0.006) and 84% (p < 0.05). Our data are questioning the rationale for empirical repetitive protamine sulphate administration when so-called "heparin rebound" after cardiac surgery is suspected, since protamine sulphate in excess of heparin may impair the platelet GPIb-vWF interaction necessary for normal haemostasis.