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1.
Thrombin-generation and activation of platelets during percutaneous coronary intervention (PCI) play a key role for early thrombotic events. Heparin and bivalirudin are approved anticoagulants for PCI. We examined the specific effects of these anticoagulants on platelet adhesion and aggregation under high shear conditions, and the presence of excess thrombin. To simulate in vivo conditions that may precipitate a bleeding/thrombotic event, we added thrombin in vitro to blood samples from 89 stable patients who had been randomly assigned to receive heparin or bivalirudin for elective PCI and examined thrombin-inducible platelet adhesion and aggregation under high shear conditions. Platelet adhesion increased by 10% of baseline with heparin, but decreased by 20% with bivalirudin (p=0.0047). Thrombin-inducible platelet adhesion and size of aggregates was equally inhibited by heparin and bivalirudin. Thus, under high shear conditions and excessive thrombin generation as they occur in atherosclerotic vascular compartments and acute vascular syndromes, heparin and bivalirudin inhibit thrombin-induced platelet adhesion and aggregation to a similar extent, while they have opposite effects on platelet adhesion in the absence of thrombin. 相似文献
2.
Phosphodiesterase (PDE)3 inhibitors exert potent antiplatelet effects through maintaining elevated intracellular cyclic adenosine monophosphate levels, but do not prolong bleeding time. To resolve this discrepancy, we hypothesised that PDE3 inhibitors effectively suppress shear-induced platelet thrombus formation initiated by the interaction of the platelet receptor GPIb/V/IX with its ligand, von Willebrand factor (VWF), since arterial thrombosis is more dependent on shear stress as compared with haemostatic plug formation. To test the hypothesis, we compared the in vitro effects of K-134 (a PDE3 inhibitor), tirofiban (a GPIIb/IIIa inhibitor) and acetylsalicylic acid (ASA) on ristocetin-induced platelet aggregation and platelet thrombus formation on VWF or collagen surfaces under flow conditions. K-134 inhibited GPIIb/IIIa-dependent platelet aggregation to the same extent as tirofiban and more potently than ASA. Likewise, K-134 and tirofiban effectively inhibited stable platelet thrombus formation (platelet firm adhesion and subsequent aggregation) on the VWF or collagen surface under high shear, but ASA only inhibited aggregation. Notably, inhibition by K-134 became evident only when a low concentration of PGE1 was present. These inhibitors did not block shear-induced initial platelet contact with VWF via GPIb/V/IX. In contrast, under low shear, the inhibitory effects of K-134 on platelet aggregation on the collagen surface were lower than tirofiban or ASA. The observed shear-dependent suppression of platelet thrombus formation by PDE3 inhibitor in the presence of low levels of adenylate cyclase stimulator may contribute to high therapeutic benefit with low risk of bleeding. 相似文献
3.
Ekmekçi H Işler I Sönmez H Gürel C Ciftçi O Ulutin T Kökoğlu E Domaniç N Dirican A 《Thrombosis research》2006,117(3):249-254
INTRODUCTION: Coronary thrombosis is an important determinant of prognosis in patients with acute coronary syndromes. Fibronectin is also found in platelets within the alpha secretory granules and secreted following platelet stimulation by a variety of agonist. Available data suggest that expression of platelet fibronectin on the cell surface may indicate a role in platelet aggregation and adhesion to fibrin thrombi and connective tissue. Clear evidence has emerged that a concerted action of nitric oxide (NO) generated by either endothelial or platelet NO synthases regulates platelet activation, causing inhibition of adhesion and aggregation. The aim of the present study was determining and correlating the serum total NO (NOx), platelet fibronectin and ADP-induced platelet aggregation levels in coronary artery disease (CAD) patient subgroups. MATERIALS AND METHODS: A total of 43 coronary artery disease patients were included in this study. Peripheral blood samples from patients with coronary artery disease were obtained from the Department of Cardiology. Platelet aggregation tests with adenosine diphosphate (ADP) were analyzed by using aggregometer. Platelet fibronectin concentrations were determined by enzyme-linked immunosorbent assay (ELISA). Serum total nitric oxide (NOx) levels were determined by colorimetric method. RESULTS: In patients with double-vessel disease, platelet fibronectin levels were found to be significantly higher than no-vessel disease (p<0.001), single-vessel disease (p<0.01) and triple-vessel disease (p<0.001). In addition, in patients with single-vessel disease platelet fibronectin levels significantly higher than no-vessel disease (p<0.05). We could not find any significant differences in ADP-induced platelet aggregation and serum NOx values between CAD patient subgroups. There was a positive correlation between platelet fibronectin levels and severity of disease (r=0.315, p<0.05). 相似文献
4.
Angiolillo DJ Bernardo E Palazuelos J Desai B Weisberg I Alfonso F Guzman LA Hernández-Antolin R Zenni MZ Macaya C Fernandez-Ortiz A Bass TA 《Thrombosis and haemostasis》2008,99(1):161-168
The currently recommended maintenance dose of clopidogrel is often associated with inadequate platelet inhibition, suggesting the need for a higher dose. The aim of this pilot study was to assess the functional impact of a high (150 mg/day) maintenance dose of clopidogrel in patients undergoing elective percutaneous coronary intervention (PCI). This is a prospective, randomized, platelet function study which was performed in elective PCI patients assigned to treatment with either a 75 mg (n = 20) or 150 mg (n = 20) daily maintenance dose of clopidogrel for 30 days; afterwards, all patients resumed standard dosing. Platelet aggregation was performed using light transmittance aggregometry following 20 microM and 5 microM adenosine diphosphate (ADP) stimuli 30 days after randomization and 30 days after resuming standard dosing. Patients treated with 150 mg/day clopidogrel had lower 20 microM ADP-induced platelet aggregation compared to patients on 75 mg/day (52.1 +/- 9% vs. 64.0 +/- 8%; p < 0.001; primary endpoint). The dose-dependent effect was confirmed by the absolute and relative increase in platelet aggregation after resuming standard dosing (p < 0.001). No changes were observed in patients randomized to standard dosing. Parallel findings were observed following 5 microM ADP stimuli for all assessments. A broad variability in clopidogrel-induced antiplatelet effects was observed irrespective of dosing. In conclusion, a 150 mg/day maintenance dose regimen of clopidogrel is associated with reduced platelet reactivity and enhanced platelet inhibition compared to that achieved with the currently recommended 75 mg/day in patients undergoing elective PCI. 相似文献
5.
Dong JF Cruz MA Aboulfatova K Martin C Choi H Bergeron AL Martini SR Kroll MH Kent TA 《Thrombosis and haemostasis》2008,99(3):586-593
Mg (++) regulates endothelial functions and has anti-inflammatory effects. Its effects on thrombosis have been demonstrated, but the mechanism remains poorly understood. We investigated the roles of MgSO(4) in regulating the release and cleavage of the prothrombotic ultra-large (UL) von Willebrand factor (VWF) and VWF-mediated platelet adhesion and aggregation. Washed platelets were perfused over cultured endothelial cells from human umbilical cord veins under a shear stress of 2.5 dyn/cm(2). Release and cleavage of ULVWF by ADAMTS-13 was measured in the absence or presence of physiological or therapeutic levels of MgSO(4). Whole blood or plasma-free reconstituted blood was perfused over immobilized collagen to measure the effect of MgSO(4) on platelet adhesion and aggregation. Also studied were the effects of MgSO(4) on ristocetin-induced platelet aggregation andVWF-collagen interaction. Maintenance of endothelial integrity required physiological levels of MgSO(4), but exogenous MgSO(4) showed no additional benefits. Exogenous MgSO(4) significantly enhanced the cleavage of the newly released ULVWF strings by ADAMTS-13 and markedly reduced platelet aggregation on immobilized collagen under flow conditions. This effect is likely to be mediated through VWF as Mg(++) partially inhibited ristocetin-induced platelet aggregation and VWF binding to collagen. MgSO(4) is critical for maintaining endothelial integrity and regulates ULVWF proteolysis and aggregation under flow conditions. These results provide a new insight into additional mechanisms involved with magnesium therapy. 相似文献
6.
Takahashi S Ushida M Komine R Shimizu A Uchida T Ishihara H Shibano T Watanabe G Ikeda Y Murata M 《Thrombosis research》2007,119(4):517-524
INTRODUCTION: Aspirin is one of the most effective antiplatelet agents and is now commonly used to prevent vascular events. In some patients, however, recurrent vascular events have been demonstrated despite aspirin therapy. Our objective was to characterize individuals showing poor response to in vitro effect of aspirin, using PFA-100. METHODS: One hundred sixty-eight healthy male subjects were analyzed. We assessed platelet function tests, including PFA-100, whole blood aggregation, and optical platelet aggregation. Also measured were hemostatic and other parameters including von Willebrand factor (VWF:Ag), VWF ristocetin cofactor activity (VWF:RCo), soluble vascular adhesion molecule-1 (sVCAM-1), high sensitive C-reactive protein (hs-CRP), and adiponectin. Poor responders were defined as having a collagen/epinephrine-induced closure time (CEPI-CT) under 250 s with PFA-100 when incubated with 10 microM aspirin, whereas good responders were defined as having a CEPI-CT of more than 250 s. RESULTS AND CONCLUSIONS: PFA-100 tests revealed that 40 subjects (24%) were poor responders (PR) and 128 (76%) were good responders (GR). Poor responsiveness was significantly associated with (1) higher basal platelet activities in PFA-100, as well as in whole blood aggregation and aggregometer;(2) increased level of adiponectin (8.8+/-4.1 micro g/mL [PR] vs 7.3+/-2.9 micro g/mL [GR], p=0.010);and (3) the presence of diabetes mellitus (17.5% [PR] vs 4.7% [GR], p=0.009). Importantly, whereas 24% of the subjects showed insufficient inhibition in PFA-100 when incubated with 10 microM aspirin, almost all subjects showed maximum inhibition with 30 microM aspirin. These observations suggest that higher doses of aspirin might overcome aspirin resistance. 相似文献
7.
INTRODUCTION: The marked interindividual variability in platelet inhibition even after administration of high loading doses of clopidogrel raised the question whether monitoring of antiplatelet effects in patients undergoing percutaneous coronary intervention (PCI) can improve clinical outcome. Established methods for monitoring antiplatelet drug activity such as optical aggregometry and determination of surface protein expression are not suitable for routine bedside testing. MATERIAL AND METHODS: We therefore compared the applicability of whole blood impedance aggregometry (20 micromol/L ADP) and the whole blood bedside ULTEGRA assay with ADP-cartridges (20 micromol/L) with optical aggregometry in platelet-rich plasma and determination of surface protein expression (P-Selectin and activated GPIIb/IIIa) by flow cytometry. We analyzed samples obtained from 27 patients scheduled for elective PCI who received a loading dose of 600 mg of clopidogrel. Blood samples were withdrawn before clopidogrel, before PCI and 24h thereafter. RESULTS: Platelet aggregation assessed by optical aggregometry (20 micromol/L ADP) declined from 65+/-9% (baseline) to 42+/-12% (PCI) and 45+/-13% (24h; p<0.01). Expression of surface proteins displayed a similar time course. Platelet aggregation determined by impedance aggregometry decreased from 4.6+/-4.0 Omega (baseline) to 0.1+/-0.3 Omega (PCI) and 0.5+/-1.1 Omega (24h) with no detectable residual platelet aggregation during PCI in 88% of patients. The ULTEGRA assay showed only slight changes after administration of clopidogrel. Correlation analysis between the various assays revealed significant correlations only between optical aggregometry and flow cytometry. CONCLUSIONS: The results indicate that both of the whole blood assays cannot substitute for optical aggregometry or determination of surface proteins in the assessment of clopidogrel-induced platelet inhibition. 相似文献
8.
Prevalence of clopidogrel non-responders among patients with stable angina pectoris scheduled for elective coronary stent placement 总被引:45,自引:0,他引:45
Müller I Besta F Schulz C Massberg S Schönig A Gawaz M 《Thrombosis and haemostasis》2003,89(5):783-787
Dual antiplatelet therapy with aspirin and clopidogrel decreases the rate of stent thrombosis in patients undergoing percutaneous coronary intervention (PCI). However, despite intensified antiplatelet treatment, up to 4.7% of the patients undergoing coronary stenting develop thrombotic stent occlusion, suggesting incomplete platelet inhibition due to clopidogrel resistance. We evaluated the percentage of clopidogrel non-responders among 105 patients with coronary artery disease (CAD) undergoing elective PCI. All patients were treated regularly with aspirin 100 mg/d and received a loading dose of 600 mg clopidogrel followed by a maintenance dose of 75 mg/d before PCI. Clopidogrel non-responders were defined by an inhibition of ADP (5 and 20 Mol/L) induced platelet aggregation that was less than 10% when compared to baseline values 4 h after clopidogrel intake. Semi-responders were identified by an inhibition of 10 to 29%. Patients with an inhibition over 30% were regarded as responders. We found that 5 (ADP 5 Mol/L) to 11% (ADP 20 Mol/L) of the patients were non-responders and 9 to 26% were semi-responders. Among the group of non-responders there were two incidents of subacute stent thrombosis after PCI. We conclude that a subgroup of patients undergoing PCI does not adequately respond to clopidogrel, which may correspond to the occurrence of thromboischemic complications. Point-of-care testing may help to identify these patients who may then benefit from an alternative antiplatelet therapy. 相似文献
9.
Ruggeri ZM 《Thrombosis research》2007,120(Z1):S5-S9
Von Willebrand factor (VWF) is a large multimeric glycoprotein produced in endothelial cells and megakaryocytes and present in subendothelial matrix, blood plasma and platelets. VWF mediates adhesion and aggregation of platelets at sites of vascular injury, processes that are critical for both haemostasis and thrombosis. Thrombus formation involves complex events that are influenced by different environmental conditions. Progress in understanding the structure and function of VWF and the mechanisms that underlie its interactions with platelets has led to important insight into the differentiation between normal haemostasis and pathological arterial thrombosis. The conventional view of signalling-induced platelet aggregation has recently been extended to include activation-independent aggregation. A novel mechanism has been demonstrated for initiating thrombus formation under high haemodynamic forces that involves alpha(IIb)beta(3)-independent platelet aggregation at the interface between immobilised and soluble VWF. This VWF-mediated process may be a key determinant of platelet accumulation in stenotic arteries leading to acute thrombotic occlusion. 相似文献
10.
van Lier M Verhoef S Cauwenberghs S Heemskerk JW Akkerman JW Heijnen HF 《Thrombosis and haemostasis》2008,99(6):1068-1078
Several studies have highlighted a specific role for membrane cholesterol domains in platelet signalling. Upon adhesion to von Willebrand factor (VWF) or collagen, cholesterol-rich domains (CRDs) accumulate in filopodial extensions and selectively harbour counterpart receptors (GPIb and GPVI) and associated signalling molecules. In the present study we have addressed the role of membrane cholesterol in Ca(2+) signalling and secretion during the interaction of platelets with VWF and collagen. VWF/ristocetin-induced platelet aggregation was delayed after treatment with methyl beta-cyclodextrin (mbCD), but the maximal aggregation response was not affected. Platelet spreading but not adhesion to immobilised VWF under flow was attenuated by cholesterol removal, and accompanied by moderate lowering in the spiking Ca(2+) response. On the other hand, platelet interaction with collagen was quite sensitive to cholesterol depletion. Platelet aggregation decreased after treatment with mbCD, and Ca(2+) responses were decreased, both under static and flow conditions. Cholesterol depletion affected the secondary feedback activation via release of thromboxane A(2) and ADP. The collagen-induced secretion of alpha granules and surface translocation of P-selectin and CD63 was also critically affected by cholesterol depletion. Confocal microscopy showed localization of p-Tyr at sites of contact with substrate and other platelets, where also CRDs accumulate. Our data thus reveal a more critical role for membrane cholesterol in collagen-induced than in VWF-induced Ca(2+) signalling, and furthermore support the concept that secondary activation responses are dependent on intact CRDs. 相似文献
11.
Marcucci R Cesari F Cinotti S Paniccia R Gensini GF Abbate R Gori AM 《Thrombosis research》2008,123(1):130-136
Background
The pathophysiologic mechanisms leading to residual platelet reactivity (RPR) on antiplatelet therapy, a condition high prevalent in patients with acute coronary syndrome, are not yet elucidated. In the acute phase of coronary artery disease large amounts of ultra large VWF multimers (ULVWF) are released and cleaved by the activity of ADAMTS-13.Objective
Aim of this study was to evaluate the relationships between VWF antigen (VWF:Ag) levels, collagen binding activity of VWF (VWF:CB), ADAMTS-13 and interleukin-6 (IL-6) levels in affecting platelet response to dual antiplatelet treatment.Methods
In 159 acute coronary syndrome (ACS) patients undergoing percutaneous coronary interventions we measured platelet function by platelet aggregation with two agonists [1 mM arachidonic acid (AA) and 10 μM ADP]. We defined patients with RPR those with platelet aggregation by AA > 20% and/or ADP (10 µmol) > 70%.Results
We found significantly lower ADAMTS-13 activity, elevated IL-6, VWF:Ag and VWF:CB levels in patients with RPR. A lower ADAMTS-13 activity was present in patients with VWF:Ag and VWF:CB in the upper tertile. At the multivariate analysis ADAMTS-13 activity and IL-6 were independent risk factors for RPR.Conclusion
Our results indicate that ADAMTS-13 activity and IL-6 levels independently affect RPR and suggest that, by different pathways, both are involved in the variable response to antiplatelet therapy. 相似文献12.
Effects of pretreatment with clopidogrel on platelet and coagulation activation in patients undergoing elective coronary stenting 总被引:2,自引:0,他引:2
Weltermann A Fritsch P Kyrle PA Schoenauer V Heinze G Wojta J Christ G Huber K 《Thrombosis research》2003,112(1-2):19-24
BACKGROUND: Current data suggest that pretreatment with clopidogrel (in addition to aspirin) prior to elective percutaneous coronary intervention (PCI) might be associated with a reduced incidence of subsequent adverse ischemic events. The aim of this placebo-controlled study was to find out whether an extended pretreatment period with clopidogrel before an elective PCI might confer a superior inhibition of the platelet activation and aggregation than clopidogrel given not until PCI. METHODS: Twenty patients with stable angina being already on aspirin were randomly assigned to receive the loading dose of 300 mg clopidogrel, either 24 h before or immediately after stent implantation. At several time points before and after PCI, the activation of both the platelet and the coagulation system was determined by measuring beta-thromboglobulin (beta-TG) and prothrombin fragment f1.2 (f1.2), respectively, in venous blood and in blood emerging from a microvascular injury (shed blood). RESULTS: Pretreatment with clopidogrel before PCI exhibited a slight reduction of beta-TG (from 178 to 139 ng/ml, p=0.085) and of f1.2 (from 0.81 to 0.75 nmol/l, p=0.045) in venous blood. Heparin administration (at the beginning of PCI) resulted in a 65% inhibition of ss-TG (from 10,590 to 2833 ng/ml) and 90% inhibition of f1.2 formation (from 38.7 to 4.2 nmol/l) in shed blood of patients with clopidogrel pretreatment. The extent of inhibition was, however, comparable to that observed in patients without clopidogrel pretreatment (beta-TG: from 8025 to 2812 ng/ml, 76% inhibition, p=0.47; f1.2: from 34.9 to 3.8 nmol/l, 86% inhibition, p=0.80). After PTT normalisation (6 h after PCI), levels of beta-TG and f1.2 both in venous blood and in shed blood did not differ between the two treatment regimens up to 48 h after PCI. CONCLUSION: Pretreatment with clopidogrel did not result in a pronounced inhibition of the platelet and coagulation system activation in patients on aspirin undergoing elective coronary stent implantation. 相似文献
13.
Szántó T Schlammadinger A Staelens S De Meyer SF Freson K Pareyn I Vauterin S Hársfalvi J Deckmyn H Vanhoorelbeke K 《Thrombosis and haemostasis》2007,98(1):178-185
Many polymorphisms in vonWillebrand factor (VWF) have been reported and their association with VWF plasma levels or cardiovascular diseases has been investigated. The aim of this study was to examine whether the amino acid polymorphism A/T1381 in the VWF A1-domain would affect VWF binding to platelet GPIbalpha. Sixty-one normal individuals were genotyped at the A/T1381 locus. Twenty-one A/A1381 homozygotes, 30 A/T1381 heterozygotes and 10 T/T1381 homozygotes were identified. Remarkably, when compared to VWF of A/T1381 and A/A1381 individuals, VWF of individuals carrying the T/T1381 variant showed an increased affinity for its platelet receptor GPIbalpha under static conditions, as reflected by an increased sensitivity to low concentrations of ristocetin or botrocetin. In addition, also the rVWF-T1381 demonstrated a higher affinity for GPIbalpha than rVWF-A1381. Interestingly, this enhanced affinity of the T/T variant over the A/T and A/A variant was, however, too subtle to affect platelet adhesion under physiological flow conditions, which fully corroborates the normal haemostatic phenotype of all individuals. We demonstrate that the VWF A/T1381 polymorphism plays an important role in inter-individual variability of the affinity of VWF for GPIbalpha, with T/T variants having a higher affinity than A/A and A/T variants, at least under static conditions in vitro. Further genetic linkage and association studies are necessary to establish whether the A/T1381 polymorphism could correlate with an increased risk of thrombotic events. 相似文献
14.
Ono T Kaikita K Hokimoto S Iwashita S Yamamoto K Miyazaki Y Horio E Sato K Tsujita K Abe T Deguchi M Tayama S Sumida H Sugiyama S Yamabe H Nakamura S Nakagawa K Ogawa H 《Thrombosis research》2011,128(6):e130-e136
Introduction
Carriers of reduced-function CYP2C19 allele on antiplatelet therapy show diminished platelet inhibition and higher rate of clinical risk. The purpose of this study was to determine cut-off levels of VerifyNow P2Y12 system associated with effective inhibition of on-clopidogrel platelet aggregation to predict carriers of CYP2C19 reduced-function allele among patients undergoing percutaneous coronary intervention (PCI).Materials and Methods
We enrolled 202 consecutive patients with stable coronary artery disease (CAD) undergoing PCI and treated with clopidogrel. All patients underwent CYP2C19 genotyping and measurement of residual platelet aggregation by VerifyNow system.Results
Carriers of CYP2C19 reduced-function allele constituted 131 (65%) of 202 CAD patients. Platelet inhibition measured by P2Y12 reaction units (PRU) and %inhibition was diminished in carriers compared with noncarriers (PRU: 290.0 ± 81.2 vs 217.6 ± 82.4, p < 0.001, %inhibition: 17.9 ± 17.8 vs 35.5 ± 22.8, p < 0.001, respectively). Multiple logistic regression analysis identified PRU and %inhibition as significant predictors of carrier state [odds ratio (OR) 4.95; 95% confidence interval (95%CI): 2.49 to 9.85; p < 0.001, OR 5.55; 95%CI: 2.80 to 10.99; p < 0.001, respectively]. Receiver-operating characteristic analysis showed that PRU and %inhibition were significant predictors of carrier state [area under the curve (AUC) 0.736 (95%CI: 0.664 to 0.808; p < 0.001), AUC 0.727 (95%CI: 0.651 to 0.803; p < 0.001), respectively]. The cut-off levels of PRU and %inhibition were 256 and 26.5% for the identification of carriers.Conclusions
Our results suggested that the cut-off levels of PRU and %inhibition to discriminate carriers of CYP2C19 reduced-function allele from noncarriers are potentially useful clinically to provide optimal clopidogrel therapy in patients with stable CAD undergoing PCI. 相似文献15.
Reininger AJ 《Thrombosis research》2008,122(Z4):S9-13
At sites of vascular injury, free-flowing platelets attach to the vessel wall to initiate thrombus formation in areas of high haemodynamic shear stress, a process that is critical for both haemostasis and thrombosis. This reaction is mediated by the binding of the platelet glycoprotein (GP) Iba to the A1 domain of immobilized von Willebrand factor (VWF), resulting in a complex series of events that includes platelet adhesion, activation and aggregation. Under elevated rates of shear stress these events support the formation of platelet-derived tethers and microparticles (MPs), which contain adhesive receptors and glycoproteins, and can bind to immobilized and soluble VWF, thereby facilitating thrombus formation. Understanding of the unique functional attributes of VWF has helped elucidate the interaction between VWF and platelets and its subsequent impact on thrombus formation. This interaction is multifaceted and modulated by different environmental conditions. A novel mechanism for initiating thrombus formation under high haemodynamic forces has recently been demonstrated. Results from direct real-time visualization of blood flow studies demonstrate activation-independent platelet aggregation, which occurs exclusively through binding of soluble VWF to platelet GPIb at very high shear stress, and may thus contribute to acute thrombotic occlusion. This article reviews the attributes of VWF and their impact on thrombus formation. 相似文献
16.
Introduction
VWF circulates in plasma as a series of heterogeneous multimers, mediating platelet tethering, translocation and finally adhesion to areas of injured endothelium under physiological high arterial blood flow. VWF-platelet binding requires conformational changes in VWF, which are induced by immobilization and shear. Because of unavailability of a simple flow-based measurement system, VWF activity assays are generally performed under static conditions. We describe an easily reproducible in vitro flow-chamber model using commercially available flow devices to examine VWF-collagen binding and VWF-mediated platelet adhesion under physiological flow conditions.Methods
The collagen surface of the flow-chamber was analyzed by atomic force microscopy. Collagen-bound VWF was characterized by multimer analysis and multi labelling immunofluorescence detection of exposed GPIb binding domains. Platelet adhesion was captured by time-lapse microscopy.Results
The described flow-chamber system facilitates multimer analysis of collagen-bound VWF, whereas all VWF multimers bound to collagen under physiological low to high shear rates. Multi labelling immunofluorescence detection exhibited exposed GPIb binding domains co-localized with VWF molecules. VWF-dependent platelet adhesion using time-lapse microscopy showed values comparable to experiments done with whole blood, and platelet adhesion was dependent on the VWF concentration.Conclusions
The established flow-chamber model represents an easy-to-set-up and customized tool for the characterization of VWF-binding to collagen as well as the determination of VWF-dependent platelet adhesion under defined flow conditions in real-time. 相似文献17.
Jan-Edvin Olsson 《Acta neurologica Scandinavica》1982,65(2):122-132
Routine blood analysis, platelet counts, number of circulating platelet aggregates (CPA) and platelet aggregation in vitro against adenosine-diphosphate (ADP), epinephrine and collagene were studied in 45 healthy controls, in 10 hospitalized patients with other neurological diseases than stroke and in 12 patients with transient ischemic attacks (TIA) before and after prophylactive treatment with anticoagulants (AC) or anti-platelet drugs (APD).
Except for lower hemoglobin and hematocrit levels in women, sex, smoking, oral contraceptives or pregnancy did not significantly influence the routine blood parameter. Smoking females taking oral contraceptives had an increased number of CPA and the most easily induced aggregation in vitro .
Patients with TIA had no significant differences in blood or platelet findings versus the healthy controls (except smoking females on oral contraceptives) or the non-stroke patients, even though individual patients could have high numbers of CPA and an easily induced platelet aggregation in vitro . Treatment with AC did not influence platelet function, whereas APD therapy decreased the number of CPA and inhibited the secondary platelet aggregation in vitro . 相似文献
Except for lower hemoglobin and hematocrit levels in women, sex, smoking, oral contraceptives or pregnancy did not significantly influence the routine blood parameter. Smoking females taking oral contraceptives had an increased number of CPA and the most easily induced aggregation in vitro .
Patients with TIA had no significant differences in blood or platelet findings versus the healthy controls (except smoking females on oral contraceptives) or the non-stroke patients, even though individual patients could have high numbers of CPA and an easily induced platelet aggregation in vitro . Treatment with AC did not influence platelet function, whereas APD therapy decreased the number of CPA and inhibited the secondary platelet aggregation in vitro . 相似文献
18.
Platelet reactivity in unstable coronary artery disease 总被引:2,自引:0,他引:2
Unstable coronary artery disease (CAD) might be related to obstructions of coronary blood flow by platelet aggregates. In 121 men and 43 women admitted to the coronary care unit with suspected unstable CAD, blood samples for tests of platelet function were obtained within 24 hours after admission. Platelet reactivity was tested in vitro in platelet rich plasma as the aggregability towards ADP 1 microM and collagen 1 mg/ml and as the sensitivity to prostacyclin (PSP). The levels of beta-thromboglobulin and platelet factor 4 were determined ex vivo in platelet poor plasma. Patients who developed a nontransmural myocardial infarction (n = 39) or had signs of myocardial ischemia at an exercise test performed within a week (n = 39) were considered to have unstable CAD while patients without signs of ischemia constituted the control group. In the acute phase the PSP was reduced in patients with unstable CAD without any difference between genders. The aggregability towards ADP was higher in women than men but otherwise there were no differences between groups or sexes in any other test in the acute phase. After 12 months there were no differences in PSP between the groups but women had a lower PSP than men. Thus, in the acute phase of unstable CAD, the platelet sensitivity to the inhibitory effects of prostacyclin was reduced which might contribute to the risk for further platelet aggregation, coronary occlusion and myocardial infarction. 相似文献
19.
Reininger AJ Agneskirchner J Bode PA Spannagl M Wurzinger LJ 《Thrombosis and haemostasis》2000,83(2):217-223
The c7E3 Fab reduces ischemic complications in patients undergoing high-risk coronary angioplasty or atherectomy. The present study investigated how c7E3 Fab inhibition of the platelet receptor glycoprotein IIb/IIIa and the endothelial vitronectin receptor affected platelet adhesion to endothelium and surface adsorbed fibrinogen under flow conditions. Platelet adhesion was examined using a stagnation point flow device with shear stress and shear rates up to 2.2 dynes/cm2 and 170 s(-1), respectively. Ex vivo adhesion was compared between two groups of patients with acute myocardial infarction (AMI) treated with angioplasty and stent implantation and a group of healthy controls. Only one AMI group received c7E3 Fab therapy. Patients in both groups were administered acetyl salicylic acid (ASA) and heparin. In AMI patients c7E3 Fab reduced platelet adhesion to adsorbed fibrinogen by 79% compared to AMI patients without c7E3 Fab treatment and by 74% compared to healthy controls. Thirty hours after termination of c7E3 Fab infusion adhesion had slightly recovered with an inhibition of 61% and 52% still present, respectively. Additionally, in vitro platelet adhesion to intact endothelium and to adsorbed fibrinogen was measured during superfusion with ADP stimulated platelet rich plasma of healthy controls to which c7E3 Fab was added at a final concentration fc of 20 microg/ml. In spite of ADP stimulation c7E3 Fab completely blocked platelet adhesion to adsorbed fibrinogen and, moreover, to intact endothelium. Preincubation of endothelial cells with c7E3 (fc = 20 [microg/ml) blocked adhesion of ADP-stimulated platelets by approximately 50%. Apart from the inhibition of platelet aggregation, c7E3 Fab added in vitro and given therapeutically in patients effectively blocks platelet adhesion to components of the injured as well as intact vessel wall under stagnation point flow conditions. 相似文献
20.
Jilma-Stohlawetz P Gorczyca ME Jilma B Siller-Matula J Gilbert JC Knöbl P 《Thrombosis and haemostasis》2011,105(3):545-552
Thrombotic thrombocytopenic purpura (TTP) can cause severe organ damage due to enhanced platelet aggregation by ultra-large von Willebrand factor (VWF) multimers. Thus inhibition of VWF by the anti-VWF ARC1779 might potentially be beneficial for TTP patients. This prospective trial tested the safety, pharmacokinetics and pharmacodynamics of the anti-VWF aptamer ARC1779 added to plasma exchange therapy (PEX) in patients with acute TTP. Seven patients received bolus primed continuous i.v. infusions of ARC1779 (1-2 μg/kg/min) in addition to PEX until remission of TTP was induced or for 14 days. Mean steady state ARC1779 plasma concentrations of 9.9 μg/ml reduced VWF activity to 5% (mean baseline activity was 125% in TTP patients compared to a reference plasma). PEX reduced ARC1779 levels by 50%, but steady state concentrations were restored rapidly with a mini-bolus. After discontinuation of PEX, ARC1779 alone further increased platelet counts in one patient. Stopping ARC1779 was associated with an immediate drop of platelet counts in this patient. This suggests that ARC1779 can block the progression of TTP in patients with severe ADAMTS13 is deficiency. ARC1779 was generally well tolerated without any signs of bleeding. Pharmacokinetics and pharmacodynamics of ARC1779 were well predictable and in agreement with those observed in a previous trial with healthy volunteers. Based on its mechanism of action and the observed effect on platelet counts, ARC1779 used as an adjunctive to PEX may help accelerate recovery from organ dysfunction. 相似文献