Decreased Platelet Deposition Research Articles

Effect of Diminazene Aceturate, an ACE2 activator, on platelet CD40L signaling induced glial activation in rat model of hypertension

Hypertension causes platelet activation and adhesion in the brain resulting in glial activation and neuroinflammation. Further, activation of Angiotensin-Converting Enzyme 2/Angiotensin (1–7)/Mas Receptor (ACE2/Ang (1–7)/MasR) axis of central Renin-Angiotensin System (RAS), is known to reduce glial activation and neuroinflammation, thereby exhibiting anti-hypertensive and anti-neuroinflammatory properties. Therefore, in the present study, the role of ACE2/Ang (1–7)/MasR axis was studied on platelet-induced glial activation and neuroinflammation using Diminazene Aceturate (DIZE), an ACE2 activator, in astrocytes and microglial cells as well as in rat model of hypertension. We found that the ACE2 activator DIZE, independently of its BP-lowering properties, efficiently prevented hypertension-induced glial activation, neuroinflammation, and platelet CD40-CD40L signaling via upregulation of ACE2/Ang (1–7)/MasR axis. Further, DIZE decreased platelet deposition in the brain by reducing the expression of adhesion molecules on the brain endothelium. Activation of ACE2 also reduced hypertension-induced endothelial dysfunction by increasing eNOS bioavailability. Interestingly, platelets isolated from hypertensive rats or activated with ADP had significantly increased sCD40L levels and induced significantly more glial activation than platelets from DIZE treated group. Therefore, injection of DIZE pre-treated ADP-activated platelets into normotensive rats strongly reduced glial activation compared to ADP-treated platelets. Moreover, CD40L-induced glial activation, CD40 expression, and NFкB-NLRP3 inflammatory signaling are reversed by DIZE. Furthermore, the beneficial effects of ACE2 activation, DIZE was found to be significantly blocked by MLN4760 (ACE2 inhibitor) as well as A779 (MasR antagonist) treatments. Hence, our study demonstrated that ACE2 activation reduced the platelet CD40-CD40L induced glial activation and neuroinflammation, hence imparted neuroprotection.

A Commercial Nonbinding Surface Effectively Reduces Fibrinogen Adsorption but Does Not Prevent Platelet Adhesion to Fibrinogen.

A commercial nonbinding surface effectively prevents protein adsorption; however, the platelet phenotype on this surface has yet to be defined. This study evaluates platelet adhesion and adsorption of several plasma/extracellular matrix (ECM) proteins to the nonbinding surface compared to other commonly used nontreated and high-binding surfaces. Platelet adhesion to uncoated microplates and those coated with fibrinogen or collagen is quantified by colorimetric assay. The binding capacity of the examined surfaces for plasma/ECM proteins is evaluated by measuring the relative and absolute protein adsorption. Compared to other surfaces, the nonbinding surface effectively prevents platelet adsorption, i.e. by 61-93% (Enzyme-Linked Immunosorbent Assay, ELISA), and reduces platelet adhesion, i.e. by 92%, when not coated with any protein. The nonbinding surface also decreases platelet deposition on collagen (up to 31%), but not fibrinogen. The nonbinding surface seems to be more of a low-fouling than nonfouling material, as it is able to reduce fibrinogen adsorption but not prevent platelet adhesion to fibrinogen. This feature should be considered when using the nonbinding surface for in vitro platelet testing.

The effect of Bruton's tyrosine kinase inhibitor ibrutinib on atherothrombus formation under stenotic flow conditions

BackgroundBruton's kinase (Btk) is critical for collagen-triggered platelet signal transduction. The Btk inhibitor ibrutinib has been shown to selectively block platelet adhesion to atherosclerotic plaque material under laminar arterial flow. However, this has not been studied under a shear gradient, which is characteristic for atherothrombosis. ObjectiveTo determine the effect of ibrutinib treatment on in vitro thrombus formation on collagen and atherosclerotic plaque material in the absence or presence of a shear gradient. MethodsBlood was obtained from patients with chronic lymphocytic leukemia, mantle-cell lymphoma and Waldenström macroglobulinemia with and without ibrutinib treatment and perfused through a microfluidic channel with(out) 60% stenosis over Horm type I collagen or human atherosclerotic plaque homogenate. ResultsAt a constant shear rate of 1500 s−1, platelet deposition was significantly decreased in blood from haematological malignancy patients treated with ibrutinib as compared to untreated patients, on atherosclerotic plaque material but not on collagen. However, thrombus size, stability, and height, were reduced on both plaque material and collagen. An increase in shear rate up to 3900 s−1, as induced by 60% stenosis, resulted in decreased platelet deposition and thrombus parameters on plaque material but not on collagen when compared to a laminar shear of 1500 s−1. Ibrutinib treatment decreased platelet deposition and thrombus parameters even further around the stenosis. ConclusionTreatment of patients with haematological disorders with the Btk inhibitor ibrutinib reduces in vitro platelet deposition, thrombus size and contraction on human atherosclerotic plaque around a stenosis when compared to patients not receiving ibrutinib.

Evaluation of the Antihemostatic and Antithrombotic Effects of Lowering Coagulation Factor VII Levels in a Non-human Primate.

Tissue factor (TF) and factor (F) VII, components of the extrinsic pathway of blood coagulation, are essential for hemostatic plug formation in response to injury; less clear are their roles in propagating thrombosis, as observational data in humans with congenital FVII deficiency suggests persistent thrombotic and bleeding risk even at significantly decreased FVII levels. We aimed to define the contribution of FVII to thrombus formation and hemostasis using a non-human primate model. We treated baboons with a FVII antisense oligonucleotide (ASO) and measured platelet and fibrin deposition inside and distal to collagen- or TF-coated vascular grafts. We assessed hemostasis by measuring bleeding time (BT) and prothrombin time (PT). Enoxaparin and vehicle treatments served as controls. FVII-ASO treatment reduced FVII levels by 95% and significantly increased both the PT and BT. Lowering FVII levels did not decrease platelet deposition in collagen- or TF-coated grafts, in thrombi distal to the grafts, or fibrin content of either collagen- and TF-coated grafts. Lowering FVII levels were associated with a modest 25% reduction in platelet deposition at 60min in the distal thrombus tail of TF-coated grafts only. FVII inhibition by way of ASO is feasible yet significantly impairs hemostasis while only exhibiting antithrombotic effects when thrombosis is initiated by vessel wall surface-associated TF exposure.

Dual antiplatelet and anticoagulant (APAC) heparin proteoglycan mimetic with shear-dependent effects on platelet-collagen binding and thrombin generation

Heparin proteoglycans (HEP-PGs) carry standard heparin-mediated anticoagulant properties as well as novel antiplatelet functions, a combination that may be significant for targeting multiple pathways in a single therapy. Recent work developing semisynthetic HEP-PG mimetics has shown promising results also in vivo, however flow conditions in vitro that replicate in vivo hemodynamics have not been reported. In this work, we present several assays (platelet calcium mobilization, aggregometry, microfluidic tests at venous and arterial hemodynamics) to characterize specific mechanistic effects of dual antiplatelet and anticoagulant (APAC) constructs as mimetics of HEP-PGs. Three APACs with different conjugation levels of heparin chains (CL10, CL18, HICL) were shown to decrease platelet deposition to collagen surfaces in PPACK-treated whole blood at venous shear rate (200 s−1). FXIIa-inhibited whole blood (CTI: corn trypsin inhibitor, 40 μg/mL) perfused over collagen/tissue factor showed reduced both platelet and fibrin deposition when treated with APACs. IC50 values for platelet and fibrin inhibition were calculated for each molecule at venous shear rate. Increasing the shear rate to arterial flows (1000 s−1) and using APAC as the sole anticoagulant, resulted in a more potent antiplatelet effect of APAC, suggesting an added effect on von Willebrand Factor (vWF) function. Additionally, APAC caused an inhibition of calcium mobilization specific to thrombin and collagen stimulation and a dose-dependent reduction in collagen-mediated platelet aggregation. Understanding the sensitivity of APAC activity to shear rate, platelet signaling and procoagulant pathways is important for applications in which APAC administration may have beneficial therapeutic effects.

Hypoxia impairs agonist-induced integrin \u03b1IIb\u03b23 activation and platelet aggregation

Under ischemic conditions, tissues are exposed to hypoxia. Although human physiology, to a certain extent, can adapt to hypoxic conditions, the impact of low oxygen levels on platelet function is unresolved. Therefore, we explored how reduction of atmospheric oxygen levels to 1% might affect agonist-induced aggregation and static adhesion of isolated human platelets. We uncovered that isolated, washed human platelets exposed to hypoxic conditions show reduced thrombin receptor-activating peptide-6 (TRAP-6) and convulxin-induced aggregation. Of note, this hypoxia-triggered effect was not observed in platelet-rich plasma. Independent of the agonist used (TRAP-6, ADP), activation of the platelet fibrinogen receptor integrin αIIbβ3 (GPIIbIIIa, CD41/CD61) was strongly reduced at 1% and 8% oxygen. The difference in agonist-induced integrin αIIbβ3 activation was apparent within 5 minutes of stimulation. Following hypoxia, re-oxygenation resulted in the recovery of integrin αIIbβ3 activation. Importantly, platelet secretion was not impaired by hypoxia. Static adhesion experiments revealed decreased platelet deposition to fibrinogen coatings, but not to collagen or vitronectin coatings, indicating that specifically the function of the integrin subunit αIIb is impaired by exposure of platelets to reduced oxygen levels. Our results reveal an unexpected effect of oxygen deprivation on platelet aggregation mediated by the fibrinogen receptor integrin αIIbβ3.

Recombinant factor VIIa addition to haemophilic blood perfused over collagen/tissue factor can sufficiently bypass the factor IXa/VIIIa defect to rescue fibrin generation.

Factor VIII (FVIII) or factor IX (FIX)-deficient haemophilic patients display deficits in platelet and fibrin deposition under flow detectable in microfluidics. Compared to fibrin generation, decreased platelet deposition in haemophilic blood flow is more easily rescued with recombinant factor VIIa (rFVIIa), whereas rFVIIa requires FXIIa participation to generate fibrin when tissue factor (TF) is absent. Perfusion of haemophilic whole blood (WB) over collagen/TF surfaces was used to determine whether rFVIIa/TF was sufficient to bypass poor FIXa/FVIIIa function in blood from patients with haemophilia A and B. Whole blood treated with high-dose corn trypsin inhibitor (40 μg mL-1 ) from seven healthy donors and 10 patients was perfused over fibrillar collagen presenting low or high TF (TFlow or TFhigh ) at wall shear rate of 100 s-1 . With WB from healthy controls, platelet deposition and fibrin accumulation increased as TF increased. Factor-deficient WB (1-3% of normal) displayed striking deficits in platelet deposition and fibrin formation at either TFlow or TFhigh . In contrast, mildly factor-deficient WB (14-32%) supported fibrin formation under flow on TFhigh /collagen. With either TFlow or TFhigh , exogenously added rFVIIa (20 nm) increased platelet deposition and fibrin accumulation in WB from factor-deficient patients (1-3% of normal) to levels commensurate with untreated healthy WB. The absence of FIXa/FVIIIa in patients with severe haemophilia results in deficits in fibrin formation that cannot be rescued by wall-derived TF ex vivo. The effects of rFVIIa on platelet adhesion and rFVIIa/TF can act together to reinforce thrombin generation, platelet deposition and fibrin formation under flow.

The Influence of Different Operating Conditions on the Blood Damage of a Pulsatile Ventricular Assist Device.

Because of pulsatile blood flow's benefit for myocardial recovery, perfusion of coronary arteries and end organs, pulsatile ventricular assist devices (VADs) are still widely used as paracorporeal mechanical circulatory support devices in clinical applications, especially in pediatric heart failure patients. However, severe blood damage limits the VAD's service period. Besides optimizing the VAD geometry to reduce blood damage, the blood damage may also be decreased by changing the operating conditions. In this article, a pulsatile VAD was used to investigate the influence of operating conditions on its blood damage, including hemolysis, platelet activation, and platelet deposition. Three motion profiles of pusher plate (sine, cosine, and polynomial), three stroke volumes (ejection fractions) (56 ml [70%], 42 ml [52.5%], and 28 ml [35%]), three pulsatile rates (75, 100, and 150 bpm), and two assist modes (copulsation and counterpulsation) were implemented respectively in VAD fluid-structure interaction simulations to calculate blood damage. The blood damage indices indicate that cosine motion profile, higher ejection fraction, higher pulsatile rate, and counterpulsation can decrease platelet deposition whereas increase hemolysis and platelet activation, and vice versa. The results suggest that different operating conditions have different effects on pulsatile VAD's blood damage and may be beneficial to choose suitable operating condition to reduce blood damage in clinical applications.

Abstract 200: Development and Proof of Concept Testing of an Ex Vivo Perfusion Chamber for Studying Platelet Adhesion to Coronary Stents Under Physiological Conditions

Background: Current-generation drug-eluting stents unblock atherosclerotic arteries, inhibit restenosis, but also render the artery prone to thrombosis by inhibiting endothelial cell function. Hence, there is strong clinical interest in developing stents that inhibit restenosis, yet are less thrombogenic. Goals of this study were to 1) develop an ex vivo method to measure platelet adhesion and aggregation on coronary stents under clinically-relevant conditions, and 2) test the hypothesis that treating metal stents with trimethylsilane (TMS) through a novel glow discharge process that coats stents with a nanoscale layer of hydrophobic plasma coating, with or without additional post-deposition glow discharge treatment with NH 3 and O 2 gas, generating elemental nitrogen and oxygen on the stent surface, inhibits platelet adhesion to stents. Methods: Bare-metal stents (BMS) made of 316L stainless steel was deployed in Masterflex tubing by inflating an over-the-wire coronary balloon within the stent. Tubing containing stents was filled with porcine indium-labeled platelets in anticoagulated whole blood and the ends were connected to create a closed circuit, which was connected to a peristaltic pump to generate unidirectional flow with shear stress of 67 s -1 , similar to that present in human coronary arteries. After 30 min of continuous flow at 37°C the tubing was disconnected and extensively rinsed to remove blood. Platelet adhesion to stents was measured with a gamma counter and by scanning electron microscopy (SEM). Results: Platelet adhesion to metal stents treated with TMS followed by NH 3 and O 2 (DC-TMS+NH 3 /O 2 ) was 56±8.2% reduced compared to BMS (n=12; P=0.003), whereas TMS treatment alone did not significantly inhibit platelet adhesion. SEM confirmed significantly decreased platelet deposition on DC-TMS+ NH 3 /O 2 stents vs. BMS. Conclusions: A novel glow discharge surface modification involving TMS, NH 3 , and O 2 gas generates stents with a bioactive surface that resists platelet adhesion under physiological conditions. Our ex vivo perfusion circuit facilitates rapid and precise comparison of stent thrombogenicity under physiological conditions, which should prove useful to other investigators developing coronary stents for clinical use.

Desmopressin (DDAVP) improves recruitment of activated platelets to collagen but simultaneously increases platelet endothelial interactions in vitro

Platelet dysfunction can cause clinically relevant bleeding. Treatment with DDAVP is advocated for this condition. DDAVP increases von Willebrand factor (VWF) on endothelial cells (ECs) and in plasma. VWF could facilitate platelet deposition on subendothelial collagen. VWF also facilitates platelet/EC interactions. Therefore DDAVP could precipitate thromboembolic events. We used a flow chamber model to study in vitro and ex vivo if DDAVP alters recruitment of platelets to EC and collagen. Resting or TRAP-activated platelets and EC were treated individually or simultaneously with 0.4 ng/ml DDAVP. Fluorophor-labeled platelets (106/ml) were resuspended in reconstituted blood and superfused across EC and collagen in an in vitro flow chamber model at arterial shear (320 s−1). Adhesion of platelets to the respective surface was recorded fluorescence microscopically and platelet covered area was assessed. TRAP significantly induced adhesiveness of platelets for collagen and EC. DDAVP pretreatment of platelets did not affect adhesiveness of resting or TRAP-activated platelets for collagen or EC. Adhesiveness of resting but not TRAP-activated platelets was induced on DDAVP-treated EC. DDAVP-conditioned EC supernatant contained vWF and significantly increased platelet deposition on collagen. Platelets from patients with clinically suspected platelet dysfunction undergoing aortic valve replacement exhibited decreased platelet deposition on collagen surfaces. In summary, our data confirm that DDAVP can induce release of platelet adhesion promoting factors from EC, which is most likely vWF. DDAVP has no direct effect on platelets. Blood samples from DDAVP-treated patients do not exhibit significantly augmented platelet deposition on collagen ex vivo. This influence of released promoting factors might cause an increase of undesirable interactions of platelets with EC.

Plant Food Delphinidin-3-Glucoside Significantly Inhibits Platelet Activation and Thrombosis: Novel Protective Roles against Cardiovascular Diseases

Delphinidin-3-glucoside (Dp-3-g) is one of the predominant bioactive compounds of anthocyanins in many plant foods. Although several anthocyanin compounds have been reported to be protective against cardiovascular diseases (CVDs), the direct effect of anthocyanins on platelets, the key players in atherothrombosis, has not been studied. The roles of Dp-3-g in platelet function are completely unknown. The present study investigated the effects of Dp-3-g on platelet activation and several thrombosis models in vitro and in vivo. We found that Dp-3-g significantly inhibited human and murine platelet aggregation in both platelet-rich plasma and purified platelets. It also markedly reduced thrombus growth in human and murine blood in perfusion chambers at both low and high shear rates. Using intravital microscopy, we observed that Dp-3-g decreased platelet deposition, destabilized thrombi, and prolonged the time required for vessel occlusion. Dp-3-g also significantly inhibited thrombus growth in a carotid artery thrombosis model. To elucidate the mechanisms, we examined platelet activation markers via flow cytometry and found that Dp-3-g significantly inhibited the expression of P-selectin, CD63, CD40L, which reflect platelet α- and δ-granule release, and cytosol protein secretion, respectively. We further demonstrated that Dp-3-g downregulated the expression of active integrin αIIbβ3 on platelets, and attenuated fibrinogen binding to platelets following agonist treatment, without interfering with the direct interaction between fibrinogen and integrin αIIbβ3. We found that Dp-3-g reduced phosphorylation of adenosine monophosphate-activated protein kinase, which may contribute to the observed inhibitory effects on platelet activation. Thus, Dp-3-g significantly inhibits platelet activation and attenuates thrombus growth at both arterial and venous shear stresses, which likely contributes to its protective roles against thrombosis and CVDs.

Anthocyanins Inhibit Platelet Activation and Attenuate Thrombus Growth In Both Human and Murine Thrombosis Models

AbstractAbstract 3197 Background:Thrombosis and cardiovascular diseases (CVDs) result from blood vessel occlusion by inappropriately activated platelets. They are the leading causes of morbidity and mortality worldwide. Anthocyanins are major phytochemicals abundant in plant food and have been shown to play a protective role against CVDs. Our previous studies have demonstrated that anthocyanins are antioxidative and prevent inflammation (J Biol Chem. 2005; 280:36792-01; Arterioscler Thromb Vasc Biol. 2007; 27:519-24), which may indirectly affect platelet function. It has also been reported that anthocyanins affect platelet activities in whole blood and platelet rich plasma (PRP). However, the direct effects of anthocyanins on platelet function and thrombus formation have not been studied. Methods:Here we investigated the effects of anthocyanins on thrombosis using purified platelets as well as several thrombosis models in vitro and in vivo. Cyaniding-3-gulucoside (Cy-3-g) and delphinidin-3-glucoside (Dp-3-g), the two predominantly bioactive compounds of anthocyanin preparations, were prepared from Polyphenol AS Company in Norway. Purified gel-filtered platelets and PRP from healthy human volunteers and C57BL/6J mice were incubated at 37°C for 10 minutes with different concentrations (0.5μM, 5μM and 50μM) of Cy-3-g, Dp-3-g or PBS buffer as a control. Platelet aggregation was assessed by aggregometry using 5μM ADP, 10μg/ml collagen, or 100μM thrombin receptor activating peptide (TRAP; AYPGKF) as agonists. Platelet adhesion and aggregation were assessed in response to an immobilized collagen matrix in an ex vivo perfusion chamber at both high (1800 s-1) and low (600 s-1) shear rates. The expression of activated GPIIbIIIa was determined via PAC-1 monoclonal antibody in flow cytometry. Lastly, the effects of anthocyanins on thrombus formation in C57BL/6J mice were assessed using a FeCl3-induced intravital microscopy thrombosis model. Results:Both Cy-3-g and Dp-3-g significantly inhibited platelet aggregation induced by collagen and TRAP in gel-filtered platelets, and inhibited aggregation induced by ADP, TRAP and collagen in human and mouse PRP. These inhibitory functions were observed at Cy-3-g and Dp-3-g doses as low as 0.5μM. Cy-3-g and Dp-3-g also reduced the surface expression of activated GPIIbIIIa on resting human platelets in a dose-dependent manner. These compounds also markedly reduced platelet adhesion and aggregation in perfusion chamber assays at both low and high shear rates. Using intravital microscopy, we further demonstrated that Cy-3-g and Dp-3-g decreased platelet deposition, destabilized thrombi, and prolonged the time required for thrombus formation and vessel occlusion. Conclusions:our data clearly demonstrated for the first time that anthocyanin compounds directly inhibited platelet activation, adhesion and aggregation, as well as attenuated thrombus growth at both arterial and veinous shear stresses. These effects on platelets likely contribute to the protective effects of anthocyanins against thrombosis and CVDs. Disclosures:No relevant conflicts of interest to declare.

Simple surface modification of a titanium alloy with silanated zwitterionic phosphorylcholine or sulfobetaine modifiers to reduce thrombogenicity

Thrombosis and thromboembolism remain problematic for a large number of blood contacting medical devices and limit broader application of some technologies due to this surface bioincompatibility. In this study we focused on the covalent attachment of zwitterionic phosphorylcholine (PC) or sulfobetaine (SB) moieties onto a TiAl 6V 4 surface with a single step modification method to obtain a stable blood compatible interface. Silanated PC or SB modifiers (PCSi or SBSi) which contain an alkoxy silane group and either PC or SB groups were prepared respectively from trimethoxysilane and 2-methacryloyloxyethyl phosphorylcholine (MPC) or N-(3-sulfopropyl)- N-(methacryloxyethyl)- N, N-dimethylammonium betaine (SMDAB) monomers by a hydrosilylation reaction. A cleaned and oxidized TiAl 6V 4 surface was then modified with the PCSi or SBSi modifiers by a simple surface silanization reaction. The surface was assessed with X-ray photoelectron spectroscopy (XPS), attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) and contact angle goniometry. Platelet deposition and bulk phase activation were evaluated following contact with anticoagulated ovine blood. XPS results verified successful modification of the PCSi or SBSi modifiers onto TiAl 6V 4 based on increases in surface phosphorous or sulfur respectively. Surface contact angles in water decreased with the addition of hydrophilic PC or SB moieties. Both the PCSi and SBSi modified TiAl 6V 4 surfaces showed decreased platelet deposition and bulk phase platelet activation compared to unmodified TiAl 6V 4 and control surfaces. This single step modification with PCSi or SBSi modifiers offers promise for improving the surface hemocompatibility of TiAl 6V 4 and is attractive for its ease of application to geometrically complex metallic blood contacting devices.

A new point-of-care method for monitoring anti-platelet therapy: Application of the cone and plate(let) analyzer

Recent studies suggest that anti-platelet agents are not equally effective in all individuals. We have developed a new method to evaluate the effect of anti-platelet drugs using the cone and plate(let) analyzer (CPA) test. The method is based on the ability of activators to reduce platelet adhesion under flow conditions. Treatment of a blood sample with arachidonic acid (AA) or ADP in vitro significantly decreased platelet deposition to a surface coverage (SC) of 2.1+/-0.4 and 1.3+/-0.6%, respectively, compared with the basic SC of 12.3+/-6.8%. The effect of AA was prevented by aspirin (SC 8.1+/-3.8%) and that of ADP was reduced by 2-methylthio-AMP, a P2Y12 ADP receptor inhibitor (SC 4.8+/-2.0%). Pre-incubation with AA of whole blood samples from untreated healthy volunteers resulted in a marked decline of SC (from SC 9.8+/-2.2 to 0.6+/-0.3%). In contrast, in volunteers treated with 100, 300, and 500 mg aspirin per day, AA (but not ADP) decreased SC only to 3.5+/-1.3, 4.4+/-1.7, and 4.1+/-2.0%, respectively (P<0.001 versus SC with AA before treatment). A good correlation was observed between the modified CPA and aggregometry (R2=0.55). In conclusion, the modified CPA test is a useful tool to evaluate the efficacy of anti-platelet therapy.

High Rate of Aspirin Resistance among Patients Undergoing Per-Cutaneous Intervention for Unstable Angina Is Associated with Clinical Outcome.

The response to anti-platelet therapy may affect clinical outcome in patients with ischemic heart disease. We prospectively investigated the response to aspirin and clopidogrel in unstable angina patients undergoing per-cutaneous intervention (PCI). Platelet function was analyzed using an aggregometer and the Cone and Platelet Analyzer (CPA) tests. In the CPA test, preincubation of blood samples with sub-optimal concentrations of arachidonic acid (AA) or ADP results in decreased platelet deposition under flow. The test was used to evaluate platelet responsiveness to aspirin and clopidogrel. Preliminary studies have demonstrated that this method is useful in detecting the response to aspirin in healthy volunteers. All patients (87) received aspirin treatment prior to hospitalization. On admission, the response to aspirin as tested by the CPA method was unexpectedly low, surface coverage (SC) with AA 2.2±0.2% (mean ± SE), with ADP 1.7±0.2%, reflecting a response rate of 26.9%. Comparable results were obtained using aggregometry, with 33.9±2.6% maximal aggregation in response to AA and 60.7±3.1% maximal aggregation in response to ADP, reflecting a response rate to aspirin of 33.3%. On the second day, after receiving 300 mg clopidogrel, the CPA assay with ADP revealed a significant response to the drug (SC 4.1±0.4%). Moreover, testing with AA on the second day demonstrated an increased response rate to aspirin (SC 4.6±0.3%), suggesting a cooperative effect of the two drugs. These results reflect response rate to aspirin of 75.4% on the second day. Aggregometry testing on the second day revealed an increased response rate up to 84.0% (maximal aggregation with AA 18.3±2.2% and with ADP 23.3±2.2%). A sub-group of 50 patients examined 6 months after the hospitalization demonstrated that the high response rate to aspirin (90%, SC 6.1±0.6%) was preserved nevertheless clopidogrel treatment had been discontinued. At the same time, according to aggregometry with AA, only 68% response rate to aspirin was observed (25.5±3.8% maximal aggregation). The response to ADP in both assays returned to the control level (SC 2.2±0.2%; maximal aggregation 70.5±3.1%), reflecting the fact that all tested patients were off clopidogrel therapy at this point. A prospective clinical evaluation revealed that 7 out of 8 patients who required additional PCI (balloon or stent) during the first 6 months after discharge were unresponsive to aspirin on the day of hospitalization as judged by both methods. In contrast, these patients except one became responders to aspirin on day 2 and after 6 months, as tested by both methods. In conclusion: a) a low response rate to aspirin was observed in patients with unstable angina undergoing PCI; b) loading dose of 300 mg of clopidogrel significantly increased the response to aspirin accordnig to both methods; c) aspirin resistance on admission might predict future complications; d) the CPA test was found useful for monitoring the effects of both aspirin and clopidogrel, and e) clopidogrel therapy before PCI in unstable angina (at least for aspirin resistant patients), may improve clinical outcome.

The influence of hemodynamics and wall biomechanics on the thrombogenicity of vein segments perfused in vitro1

This study addresses the hypothesis that exposure to peripheral arterial (ART) or coronary (COR) hemodynamics and wall biomechanics affect platelet deposition on vein segments. Intact human saphenous vein (HSV) and porcine internal jugular vein (PIJV) segments were studied under venous (VEN), ART, and COR environments using in vitro perfusion systems. Wall shear stress (tau) and circumferential wall stress (sigma(theta)) were calculated for PIJV segments. Platelet deposition was measured using a radioactive assay. PIJV ART segments exhibited a 14% increase in inner diameter over time (P < 0.05). tau, acting on PIJV ART specimens, was less at 6 h compared with time 0 (P < 0.05). sigma(theta) was lower in the VEN specimens compared with ART and COR groups (P < 0.01). Platelet deposition decreased by 40% on PIJV ART segments (P < 0.05) but increased 3.2-fold on PIJV COR segments (P < 0.05) versus VEN control segments. Platelet deposition was increased 1.75-fold in COR HSV cases versus VEN segments. These data indicate that short-term exposure to COR conditions lead to enhanced platelet deposition, whereas ART conditions decrease platelet deposition.

L-arginine improves endothelial vasoreactivity and reduces thrombogenicity after thrombolysis in experimental deep venous thrombosis.

Nitric oxide (NO) is important in regulation of platelet aggregation, endothelial function, and intravascular thrombosis. The purposes of this study were to assess the effect of thrombolysis on endothelial function in a porcine model of deep venous thrombosis (DVT) and to evaluate the effect of NO precursor l-arginine on endothelial function after thrombolytic therapy. DVT was created in bilateral iliac veins by deploying a self-expanding stent-graft that incorporated an intraluminal stenosis, from a groin approach. Five pigs underwent sham operation. After 7 days of DVT, animals were randomized to three groups: saline pulse-spray (saline group, n = 5), thrombolytic pulse-spray with tissue plasminogen activator (alteplase, 8 mg; t-PA group, n = 5), and thrombolytic pulse-spray plus intravenous l-arginine (20 mmol/L; arginine group, n = 5). At 2 weeks iliac vein patency was evaluated at venography and intravascular ultrasound scanning. NO level was determined with a chemiluminescent assay of the nitrite and nitrate metabolites (NO(x)). Thrombogenicity was evaluated with radiolabeled platelet and fibrin deposition. Veins were harvested and evaluated with light microscopy and scanning electron microscopy. Endothelial function was evaluated with organ chamber analysis. All iliac veins remained patent at 2 weeks. The luminal areas in the sham, saline, t-PA, and arginine groups were 53 +/- 23 mm(2), 14 +/- 11 mm(2), 34 +/- 19 mm(2), and 42 +/- 21 mm(2), respectively. No difference in endothelial cell structure was observed between the three treatment groups at light microscopy or scanning electron microscopy. Although no difference in fibrin deposition was noted among the three treatment groups, decreased platelet deposition occurred in the arginine group compared with the saline or t-PA groups (P <.05). The arginine group showed greater endothelial-dependent relaxation compared with the t-PA or saline groups (73% +/- 23% vs 49% +/- 18% and 32% +/- 21%; P <.05). Local NO(x) level in the arginine group was correspondingly higher compared with the saline or t-PA groups (1.8 +/- 0.3 micromol/L vs 0.3 +/- 0.05 micromol/L and 0.2 +/- 0.04 micromol/L; P <.05). NO precursor l-arginine supplementation enhances NO production at sites of venous thrombosis. Moreover, l-arginine preserves endothelial vasoreactivity and reduces platelet deposition after thrombolysis in iliac DVT. These data suggest that l-arginine may preserve endothelial function after thrombolysis and may reduce the likelihood of postthrombotic syndrome.

Inhibition of microsurgical thrombosis by the platelet glycoprotein IIb/IIIa antagonist SR121566A.

Despite major improvements in tools and significant refinements of techniques, microsurgical anastomosis still carries a significant risk of failure due to microvascular thrombosis. The key to improving the success of microvascular surgery may lie in the pharmacologic control of thrombus formation. Central to pathologic arterial thrombosis are platelets. Glycoprotein IIb/IIIa is a highly abundant platelet surface receptor that plays a major role in platelet aggregation by binding platelets to each other through the coagulation factor fibrinogen. To explore the ability of antithrombotic agents to prevent microvascular thrombosis, a rabbit ear artery model was used in which a standardized arterial injury results in predictable thrombus formation. This model was used to examine whether SR121566A, a specific and potent glycoprotein IIb/IIIa inhibitor, can successfully prevent microsurgical thrombosis. Using a coded, double-blind experimental design, 20 rabbits (40 arteries) were assigned to four treatment groups: (1) saline injection (n = 10), (2) acetylsalicylic acid 10 mg/kg (n = 10), (3) heparin 0.5 mg/kg bolus with subsequent intermittent boluses of 0.25 mg/kg every 30 minutes (n = 10), and (4) SR121566A 2 mg/kg bolus (n = 10). After vessel damage and clamp release, arteries were assessed for patency at 5, 30, and 120 minutes by the Acland refill test. Coagulation assays, in vivo bleeding times, and ex vivo platelet aggregation studies were also conducted. Scanning electron microscopy was used to examine mural thrombus composition.A significant, fourfold increase in vessel patency following administration of SR121566A over saline control (80 percent versus 20 percent patency, respectively, at 35 minutes after reperfusion, p < 0.01) was noted. This was correlated with marked inhibition of ex vivo platelet aggregation. This antiplatelet treatment did not prolong coagulation assays (mean international normalized ratio: saline, 0.66 +/- 0.04; SR121566A, 0.64 +/- 0.03; mean thromboplastin time: saline, 19.63 +/- 0.67; SR121566A, 17.87 +/- 3.27) and bleeding times (mean bleeding time: saline, 42 +/- 4; SR121566A, 48 +/- 6). Scanning electron microscopy demonstrated extensive platelet and fibrin deposition in control vessel thrombi. In contrast, thrombi from SR121566A-treated vessels demonstrated predominance of fibrin with few platelets when examined under scanning electron microscopy.Administration of SR121566A was associated with a significant increase in vessel patency, without deleterious effects on coagulation assays or bleeding times. The increase in vessel patency was correlated with inhibition of platelet aggregation and decreased platelet deposition, as demonstrated by scanning electron microscopy. Glycoprotein IIb/IIIa antagonists represent a new class of anti-platelet agents that may be suited for inhibiting microsurgical thrombosis. This study supports further investigation into the use of these agents in microsurgery.

Aurintricarboxylic Acid Attenuates Intimal Thickening after Balloon Injury of the Rabbit Aorta

Injury to the arterial wall initiates a cascade of events including platelet deposition and an increase in procoagulant activity of the vessel wall that is associated with intimal thickening and vascular wall remodeling. This study was designed to characterize the effects of aurintricarboxylic acid (ATA), an inhibitor of von Willebrand factor function, on vascular procoagulant activity and the development of intimal thickening after balloon-induced injury to the rabbit aorta. Treatment with ATA, aspirin, or the combination of agents at doses that attenuated platelet aggregation decreased platelet deposition and procoagulant activity bound to the vessel wall after injury. Treatment with ATA reduced the intimal thickening observed 2 weeks after injury. Surprisingly, aspirin treatment had no effect on intimal thickening. These data indicate that inhibition of platelet deposition, while it is able to attenuate local thrombin elaboration, is not alone sufficient to attenuate subsequent intimal thickening that occurs in response to arterial injury.

Polyethylene glycol diisocyanate decreases platelet deposition after balloon injury of rabbit femoral arteries.

Platelet deposition after angioplasty remains problematic and may contribute to intimal hyperplasia and restenosis. We proposed that polyethylene glycol diisocyanate (PEG-DISO), a polymer that rapidly forms covalent linkages with amine residues on proteins, could mask thrombogenic vascular wall proteins from platelets, thereby abrogating acute platelet deposition. To test this hypothesis, we isolated the femoral arteries of 10 New Zealand White rabbits and injured them with 3 passes of a 2F Fogarty catheter which was inserted through a distal arteriotomy. Immediately after balloon injury, (111)indium-labeled autologous platelets were infused peripherally and the injured femoral arteries were randomly treated for 1 minute with a PEG-DISO solution in one artery and a control solution of the phosphate buffered saline vehicle in the contralateral artery. Following treatment, reflow was initiated. The vessels were harvested after 1 hour and radioactivity was quantified in a gamma counter. Platelet counts were standardized by weight and expressed as platelets/mg (mean +/- SEM). Platelet deposition onto arteries treated with PEG-DISO was (1.2 +/- 0.5) x 10(6) platelets/mg compared to (5.6 +/- 4.2) x 10(6) platelets/mg onto the contralateral control arteries treated with vehicle (P < 0.005). Scanning electron micrographs of the injured vessel segment confirmed qualitatively less platelet deposition on the treated segments than on the control segments. Treatment with PEG-DISO significantly inhibited platelet deposition after vascular injury. These data support the hypothesis that treatment with PEG-DISO masks surface adhesive proteins from platelet receptors in vivo and that the resulting molecular barrier significantly reduces platelet deposition onto the damaged vessel wall for at least one hour. The formation of a molecularly thin barrier to platelet deposition may thus be a novel and effective treatment to abrogate acute intravascular thrombosis and may have value in the treatment of restenosis.