Platelet Microaggregates Research Articles

The dopamine agonism on ADP-stimulated platelets is mediated through D2-like but not D1-like dopamine receptors

Monoamines such as serotonin and epinephrine are known to be involved in platelet activation and aggregation. Dopamine is another monoamine identified in platelets, but published data about its effect on platelets and the receptors involved are controversial. In the present study, we investigated the dopamine agonism in platelets and the receptors involved in these pathways. Platelet-rich plasma (PRP) of healthy individuals was treated with agonists (ADP, epinephrine, dopamine) and various dopamine receptor and transporter antagonists such as SCH-23390, raclopride, clozapine, methylphenidate, and cocaine. Platelet activation was investigated by flow cytometry (CD62P and CD63 surface expression), optical aggregometry, and microaggregate adhesion to collagen IV in a flow chamber system. In our study, dopamine on its own had no effect on platelet activation in the different assays. However, when used in combination with ADP (10 muM), dopamine in a range of 1 to 100 muM significantly potentiated platelet microaggregate formation and adhesion to collagen under low shear flow conditions. Specific antagonists for D2-like receptors (L-741,626, raclopride, and clozapine) completely diminished the dopamine effect at selective concentrations, but not the effect of epinephrine. Neither the D1-like receptor antagonist SCH-23390 nor dopamine transporter antagonists (methylphenidate, cocaine) showed inhibitory effects on the dopamine agonism. Thus, dopamine is an ADP-dependent platelet agonist which acts via D2-like but not D1-like receptors or adrenergic receptors. Because many psychopharmacological drugs are directed to D2-like receptors, platelet dysfunction in patients being treated with such drugs may be linked to these mechanisms.

Utilidad de la citometría de flujo en la determinación de microagregados plaquetarios en pacientes con stent tratados con clopidogrel

Introduction It is not well established whether non-responder patients(NR) to clopidogrel show, as a risk factor, higher number of platelet microaggregates (PMAs) than responder patients (R). These MAPs can affect the microcirculation and is a risk factor to forming larger thrombi, therefore the present study is interesting from this point of view. Material and methods Seventy-eight acute coronary syndrome (ACS) patients (78% male, aged 62.8±12.23 years) were included in this study. These patients underwent coronary stent implantation and treated with routine medication (clopidogrel 75 mg/day and aspirin 100 mg/day), after a loading bolus of 300 mg clopidogrel. The control group was made up of 50 healthy volunteers matched for age and gender who had not been given any pharmacological treatment. Using whole blood flow cytometry, ADP-stimulated and circulating platelet CD62 expression and platelet microaggregates was determined in the entire study population. After a week of treatment the number of circulating platelet microaggregates was evaluated by flow cytometry (EPICS-XL, Beckman-Coulter, Izasa), and the number of MAPs formed ex vivo by ADP 2.5 μM activation was also evaluated . With this aim, using healthy individuals, the platelet population was selected by their forward and side scatter values and as CD61-positive events. The subpopulation that occupied the upper 5% in this region where MAPs are detected,was chosen. After the analytical conditions were established, samples were analyzed. Patients were responders, or not, depending on the CD62 expression after being activated with ADP 2.5 μM. Results Seventy-eight patients showed a higher number of circulating MAPs than controls (177±75/5000 platelets vs. 121±62/5000 platelets; P<0.001). The increase in the MAPs number by ADP activation is higher in the 50 controls than in the 51 responder patients (79±45% vs. 23±18%; P<0.001). The 27 NR patients reacted to ADP in a similar way than controls, with a increase in the number of MAPs of 62±43%, significantly higher than in R ( P<0.001). The findings of this study demonstrate the beneficial effects of clopidogrel in reducing platelet reactivity in 66% of the study patients. These results demonstrate the wide inter-individual variability in ADP response in patients treated with clopidogrel and imply that individualized monitoring of this type of patient is advisable. Conclusions The method described enables us to explore platelet microaggregates formation and may be of great help in monitoring clopidogrel efficacy in ACS patients with stent.

Flow cytometric assays for quantifying activated ovine platelets.

Ovines are a common animal model for preclinical evaluation of cardiovascular devices including heart valves, endovascular grafts, and ventricular assist devices. Biocompatibility is essential to the success of these devices; however, tools to assess biocompatibility in ovines are limited. To address this need, antibodies that bind to activated human and bovine platelets and annexin V protein were evaluated for potential cross-reactivity to activated ovine platelets. These candidate markers were incubated with stimulated and quiescent ovine whole blood, and binding to platelets was quantified by flow cytometry. Several antihuman CD62P antibodies including one polyclonal antibody, three monoclonal antibodies, and annexin V selectively bound to activated ovine platelets. An assay to quantify platelet microaggregates was also developed. The availability of assays to quantify ovine platelet activation can increase the quality of biocompatibility data obtainable during preclinical development of artificial organs in the ovine model, potentially aiding in the evaluation of design refinements to enhance device biocompatibility.

Circulating microemboli in patients with myeloproliferative disorders

Myeloproliferative disorders (MPD) are associated with an increased risk for thrombembolic events. In this study, we examined the prognostic value of transcranial Doppler (TCD) microemboli detection regarding clinical events and correlated TCD findings with results of blood cell counts and platelet flow cytometry to gain insight into the composition of circulating microemboli in these patients. In a cohort of 42 patients with MPD TCD microemboli detection was performed on a single occasion and correlated with thrombembolic events during a prospective follow up of 29.7 +/- 7.3 month. In all patients, a complete blood count and in 17 patients platelet flow cytometry were performed on the day of the TCD examination. Microembolic signals (MES) were recorded in 15 (35.7%) patients, however, without any correlation with the type of MPD, blood cell counts, or thrombembolic events [9 (21.4%)]. MES positive and negative patients did not differ regarding the levels of activated platelets, platelet microaggregates, or microparticles. We found a strong trend for higher rates of platelet-neutrophil conjugates in MES positive patients (P = 0.09). Detection of MES by TCD on a single occasion in MPD patients has only limited prognostic value. MES do not correlate with the type of MPD, nor blood cell counts. Flow cytometry suggests that MES in MPD may consist of platelet-neutrophil aggregates.

Effects of platelet glycoprotein IIb-IIIa number and glycoprotein IIIa Leu33Pro polymorphism on platelet aggregation and sensitivity to glycoprotein IIb-IIIa antagonists

We investigated the influence of glycoprotein (GP) IIIa Leu33Pro polymorphism, platelet GP IIb-IIIa number, and plasma fibrinogen concentration on platelet aggregation and antiaggregatory action of GP IIb-IIIa antagonists. Healthy volunteers with GP IIIa Pro33(−) (Leu33Leu33, n = 20) and Pro33(+) (Leu33Pro33, n = 13, and Pro33Pro33, n = 2) genotypes were included into the study. GP IIIa Leu33Pro substitution was associated with the increase of the level and rate of platelet microaggregate formation induced by GP IIb-IIIa activating antibody CRC54 (100, 200, 400 µg/ml) against the epitope within 1–100 residues of GP IIIa N-terminal part (p from 0.001 to 0.047). No significant differences were detected between parameters of platelet aggregation induced by ADP (1.25, 2.5, 5.0, 20 µM) in GP IIIa Pro33(+) and Pro33(−) donors. GP IIb-IIIa antagonist Monafram (F(ab’)2 fragment of GP-IIb-IIIa blocking antibody CRC64) (1, 2, 3 µg/ml), but not eptifibatide (50, 100, 150 ng/ml) inhibited ADP-induced aggregation slightly less efficiently in GP IIIa Pro33(+) group (p < 0.05 at 1 and 2 µg/ml Monafram). GP IIb-IIIa number (evaluated as maximal binding of 125I-labelled antibody CRC64) varied from 40.5 to 80.8 × 103 per platelet with no significant influence of GP IIIa genotype. Consistent correlations were revealed between GP IIb-IIIa quantity and the level and rate of ADP-induced aggregation (r from 0.353 to 0.583, p from <0.001 to 0.037) as well as resistance (level of residual aggregation) to both GP IIb-IIIa antagonists (r from 0.345 to 0.602, p from <0.001 to 0.042). ADP-induced aggregation was considerably increased and efficiency of GP IIb-IIIa antagonists decreased in donors with high in comparison with low GP IIb-IIIa quantity (>60 and 40–50 × 103 per platelet respectively, p < 0.01 for most tests). No correlations were observed between all tested parameters and plasma fibrinogen concentration. Our results indicate that inter-individual variability of platelet GP IIb-IIIa number significantly affects platelet aggregation and antiaggregatory effects of GP IIb-IIIa antagonists. Contribution of this factor is higher than that of GP IIIa Leu33Pro polymorphism and variations of fibrinogen concentration.

Platelet Activity, Coagulation, and Fibrinolysis During Exercise in Healthy Males

Relationships between exercise-induced activation of platelets, blood coagulation, and fibrinolysis, and the importance of thrombin for responses to exercise are not clear. Effects of thrombin inhibition on hemostatic parameters were examined in a double-blind crossover study comparing the direct thrombin inhibitor argatroban (350 microg/kg intravenous bolus followed by 25 microg/kg per minute of infusion), the indirect thrombin inhibitor enoxaparin (0.75 mg/kg, intravenous bolus), or placebo (saline) in 21 healthy males. Measurements were made at rest, before and during/after thrombin inhibitor treatment, and immediately after exhaustive exercise. At rest argatroban abolished, and enoxaparin attenuated platelet activation by thrombin, but not by adenosine diphosphate. Argatroban and, even more so, enoxaparin decreased thrombin generation (prothrombin F1+2) and the coagulation potential, and increased the fibrinolytic potential. Exercise increased circulating activated platelets (from 5.5+/-0.3 to 9.4+/-0.9x10(9)/L; P<0.001), circulating platelet-platelet microaggregates, the platelet responsiveness to in vitro stimulation, leukocyte activation (leukocyte CD11b expression and plasma elastase), and platelet-leukocyte aggregation (P<0.01 for all). Exercise increased coagulation (F1+2; P<0.01) and fibrinolysis, but did not alter the balance between them; fibrin gel permeability increased (P<0.01), probably because of release of endogenous tissue plasminogen activator from the vessel wall. Neither argatroban nor enoxaparin counteracted exercise-induced platelet or leukocyte activation. Both thrombin inhibitors augmented exercise effects on fibrinolysis. Strenuous exercise enhances platelet and leukocyte activation independently of thrombin. Exercise augments both coagulation and fibrinolysis, but the balance between them appears to be maintained. At therapeutic dosages argatroban counteracted thrombin-induced platelet activation most efficiently, whereas enoxaparin had somewhat stronger anticoagulant and profibrinolytic effects.

Preclinical biocompatibility assessment of the EVAHEART ventricular assist device: Coating comparison and platelet activation

Thromboembolism and bleeding remain significant complications of ventricular assist device (VAD) support. Increasing the amount of biocompatibility data collected during preclinical studies can provide additional criteria to evaluate device refinements, while design changes may be implemented before entering clinical use. Twenty bovines were implanted with the EVAHEART centrifugal VAD for durations from 30 to 196 days. Titanium alloy pumps were coated with either diamond-like carbon or 2-methoxyethyloylphosphoryl choline (MPC). Activated platelets and platelet microaggregates were quantified by flow cytometry, including two new assays to quantify bovine platelets expressing CD62P and CD63. Temporally, all assays were low preoperatively, then significantly increased following VAD implantation, before declining to a lower, but still elevated level over 2-3 weeks. MPC-coated VADs produced significantly fewer activated platelets after implant trauma effects diminished. Three animals receiving no postoperative anticoagulation had similar amounts of circulating activated platelets and platelet microaggregates as animals receiving warfarin anticoagulation. Two new methods to quantify bovine activated platelets using antibodies to CD62P and CD63 were characterized and applied. These measures, along with previously described assays, were able to differentiate between two biocompatible coatings and assess effects of anticoagulation regimen in VAD preclinical testing.

Blood Biocompatibility Assessment of an Intravenous Gas Exchange Device

To treat acute lung failure, an intravenous membrane gas exchange device, the Hattler Catheter, is currently under development. Several methods were employed to evaluate the biocompatibility of the device during preclinical testing in bovines, and potential coatings for the fibers comprising the device were screened for their effectiveness in reducing thrombus deposition in vitro. Flow cytometric analysis demonstrated that the device had the capacity to activate platelets as evidenced by significant increases in circulating platelet microaggregates and activated platelets. Thrombus was observed on 20 +/- 6% of the surface area of devices implanted for up to 53 h. Adding aspirin to the antithrombotic therapy permitted two devices to remain implanted up to 96 h with reduced platelet activation and only 3% of the surface covered with thrombus. The application of heparin-based coatings significantly reduced thrombus deposition in vitro. The results suggest that with the use of appropriate antithrombotic therapies and surface coatings the Hattler Catheter might successfully provide support for acute lung failure without thrombotic complications.

Platelet microaggregation inhibition in patients with acute myocardial infarction pretreated with tirofiban and relationship with angiographic and clinical outcome

The relationship between the level of platelet aggregation inhibition in patients with acute myocardial infarction and their clinical outcome is unknown. In patients with acute myocardial infarction included in the On-TIME trial and transferred to the primary percutaneous coronary intervention (PCI) center of Zwolle, who were pretreated with tirofiban on top of acetylsalicylic acid and heparin, platelet microaggregation inhibition was assessed on admission and immediately after PCI, using the Sysmex K4500 (Sysmex Corp, Kobe, Japan) platelet microaggregation measurement. The level of platelet microaggregation inhibition was compared with angiographic and clinical outcome. Patients were randomized to early prehospital initiation of tirofiban or to initiation in the catheterization laboratory. Therefore, the effect of tirofiban on platelet microaggregation inhibition could additionally be determined by measuring baseline platelet microaggregation also at entrance into the hospital. In 412 (89%) of 463 patients, platelet microaggregation inhibition was measured after receiving tirofiban. There was no difference between the 4 quartiles of the level of platelet microaggregation inhibition with regard to distal embolization, TIMI-3 flow and blush grade 3 after PCI, mean corrected TIMI frame count, ejection fraction, enzymatic infarct size, and percentage ST-segment resolution (P values .91, .97, .46, .94, .73, .33, and .72, respectively). The baseline platelet microaggregation inhibition in patients treated with tirofiban was 38% +/- 25% (mean +/- SD), and in the patients treated with placebo, 14% +/- 22% (P < .001). We found no correlation between the level of platelet microaggregation inhibition after tirofiban and outcome, whereas only a modest increase in platelet microaggregation inhibition was observed after a commonly used dose of tirofiban.

Utility of a whole blood single platelet counting assay to monitor the effects of tirofiban in patients with acute coronary syndromes scheduled for coronary intervention

This study aimed to establish the utility of a whole-blood single-platelet counting (WBSPC) assay, a measure of microaggregation, in monitoring the effects of tirofiban, comparing this with optical aggregometry (OA) and the Ultegra TRAP cartridge system (UTC), measures of macroaggregation. Fifty-nine patients with acute coronary syndrome scheduled for coronary angiography +/- angioplasty were studied. WBSPC assay (ADP 0.3-100 microM, Sysmex KX21 analyzer), OA (ADP 20 microM) and UTC were performed: before starting tirofiban; 30 min, 4 and 24 h after starting tirofiban; and 1 and 2 h after stopping tirofiban. Thirty minutes after starting tirofiban, there was substantial inhibition of platelet aggregation (40 +/- 30%; WBSPC, 2 minutes after addition of ADP 30 microM) and this remained stable at 4 and 24 h. OA (86 +/- 17%; inhibition of maximal aggregation, ADP 20 microM) and UTC (93 +/- 7%) showed marked inhibition with less inter-individual variation. There was no significant correlation between OA and UTC results (R(2) = 0.006), but fair correlation between OA and WBSPC results (R(2) = 0.37). Greater inhibition of macroaggregation (OA and UTC) was seen compared to microaggregation (WBSPC) such that WBSPC was more discriminating in the therapeutic range when macroaggregation was often completely inhibited. A WBSPC assay of platelet microaggregation shows promise for monitoring GPIIb/IIIa antagonists.

Platelets activated by collagen through the immunoreceptor tyrosine-based activation motif in the Fc receptor γ-chain play a pivotal role in the development of myocardial ischemia-reperfusion injury

Platelet activation and the formation of platelet microaggregates in coronary vessels play pivotal roles in myocardial ischemia and reperfusion injury. The Fc receptor γ-chain (FcR γ) is coexpressed with glycoprotein (GP) VI, forming a platelet collagen receptor, and the activation of platelets by collagen is closely coupled with tyrosine phosphorylation of the FcRγ. To examine the functional significance of platelet FcR γ/GPVI complex in the early phase of myocardial ischemia and reperfusion injury in mice, we performed coronary occlusion and reperfusion experiments using wild type mice and FcRγ-deficient (FcRγ –/–) mice that lack GPVI. The infarct size was significantly smaller in FcRγ –/– mice subjected to occlusion and reperfusion of the coronary artery than in control FcR γ +/+ mice. Twenty-four hours after the reperfusion, electron microscopy of the injured tissue showed substantially more platelet aggregation and occlusive platelet microthrombi in the capillaries of the damaged areas of the wild type mice than in those of the FcR γ –/– mice. Platelet Syk was scarcely activated in the FcR γ –/– mice after myocardial ischemia and reperfusion, but significantly activated in the FcR γ +/+ mice. CD11b expression on neutrophils was elevated after myocardial ischemia and reperfusion in both mouse groups, whereas myeloperoxidase activity in the injured areas was significantly lower in the FcRγ –/– mice than in the FcRγ +/+ mice. These results suggest that the collagen-induced activation of platelets through the FcR γ plays a pivotal role in the extension of myocardial ischemia-reperfusion injury. FcRγ and GPVI may be important therapeutic targets for myocardial ischemia-reperfusion injury.

Effect of doxazosin gastrointestinal therapeutic system on platelet degranulation and platelet–leukocyte microaggregate formation induced by physiologic shear stress in hypertension

Introduction In this prospective, ex vivo, single-blind study, the effect of doxazosin on platelet function was studied in patients with hypertension. Materials and methods Platelet activation by shear stress was measured in whole blood samples of 22 hypertensive patients and 22 normotensive controls, using flow cytometry. Sheared samples were evaluated for CD62 expression, microaggregate formation, and Ca 2+ mobilization. Results were collected at baseline and after 1 and 2 months of single-dose (4 mg/d) extended-release doxazosin gastrointestinal therapeutic system therapy. Results Doxazosin normalized blood pressure in hypertensive patients after 1 and 2 months of treatment. Hypertensive patients had a higher baseline percentage (mean ± SD) of degranulated platelets (CD62+) than the normotensive control group (4.14 ± 1.05 vs. 2.47 ± 0.68, P < 0.01). After 2 months of doxazosin gastrointestinal therapeutic system treatment, the percentage of CD62+ in the experimental group significantly decreased ( P < 0.05). At baseline, the number of platelet–leukocyte aggregates in vivo was greater in hypertensive patients ( P < 0.01); doxazosin did not normalize this measurement. Following shearing, platelet expression of CD62 increased significantly in the hypertensive group ( P < 0.001 vs. control). Shear stress-induced platelet activation and microaggregate formation were also greater in hypertensive patients. Intraplatelet-free calcium concentration was higher in hypertensive patients at baseline than in the normotensive group ( P < 0.001). At 2 months, doxazosin significantly reduced thrombin-stimulated Ca 2+ mobilization in hypertensive patients ( P < 0.01 vs. baseline). Conclusions Platelets from hypertensive patients are more readily activated by shear stress and demonstrate significant alterations in cytoplasmic-free calcium mobilization. Doxazosin treatment reduced blood pressure and normalized alterations in platelet function.

Red blood cell deformability and diabetic nephropathy

To the editor: In an interesting study, Brown et al1.Brown C.D. Ghali H.S. Zhao Z. et al.Association of reduced red blood cell deformability and diabetic nephropathy.Kidney Int. 2005; 67: 295-300Google Scholar found an impaired red blood cell deformability in 57 adult type 2 diabetic patients. They used a filtration technique using polycarbonate membranes with straight channels of 3 micrometer pore diameter. In patients with diabetic nephropathy they found an increased impairment in red blood cell deformability. The hypothesis that an impaired red blood cell deformability contributes to renal function decline is very attractive and may give rise to therapeutic possibilities. Many studies found an impaired red blood cell deformability in diabetic patients; others, however, did not. Years ago we tested the hypothesis that red blood cell deformability contributes to diabetic nephropathy using both ektacytometry and erythrocyte filtration, with micropores with a diameter of 5 micrometer2.Schut N.H. Van arkel E.C. Hardeman M.R. et al.No decreased erythrocyte deformability in type 1 (insulin-dependent) diabetes, either by filtration or by ektacytometry.Acta Diabetol. 1993; 30: 89-92Google Scholar. In order to imitate local circumstances in the kidney red blood cell, deformability was also measured in hyperosmolar solutions. Seventy-one insulin-dependent diabetic patients were included, 25 patients without any sign of organ damage, 21 patients with microalbuminuria, 13 patients with overt nephropathy, and 12 patients with leg ulceration. No decreased red blood cell deformability was found in any of the diabetic groups with either technique, neither did the total group of 71 diabetic patients have a lower red blood cell deformability when compared to controls. Extaerythrocytic factors, such as leukocytes, plasma fibrinogen, and platelet microaggregates may influence filtration results. I suppose the filtration technique of Brown et al was corrected for this. These results may suggest that the filtration pore size of 3 micrometers used by Brown et al was more sensitive than the 5 micrometer pores filtration technique and ektacytometry that was used by us and that the lack of difference in our patient groups reflects this lack of sensitivity. On the other hand, the question arises whether such subtle differences in red blood cell deformability, if present, contribute to nephropathy in diabetic patients.

Intracoronary formation and retention of micro aggregates of leukocytes and platelets contribute to postischemic myocardial dysfunction

Cardiac pump function and coronary regulation can be impaired after short-term ischemia. Recent studies with platelets (P) and neutrophils (PMN) yielded contradicting results about the "cellular" contribution to reperfusion injury. Isolated guinea pig hearts performing pressure-volume work were employed, external heart work (EHW), aortic flow (AF), coronary flow (CF) and heart rate (HR) serving as parameters of cardiac function. After global ischemia, human blood cells were given as bolus (1 min) during reperfusion (intracoronary hematocrit 7%). Expression of specific adhesion molecules (P: CD62P, CD41; PMN: integrin CD11b) was measured on cells before and after coronary passage (FACS analysis). Postischemic recovery of pump function was significantly reduced in hearts with blood cell application (EHW: -cells 54 +/- 14%, +cells 41 +/-12%, p <0.05). Coronary response to bradykinin and reactive hyperemia were not effected. The blood-cell dependent functional loss was partly reduced by blocking CD18 (anti-CD 18) and completely abrogated by blockage of CD41 (lamifiban). The expression of CD11b on PMN and monocytes (M) and CD62P on platelets was significantly reduced in the coronary effluent and a significant decrease of CD41 on leukocytes occurred during coronary passage after ischemia. Increases in CD41 on PMN in the presence of lamifiban demasked intracoronary formation of micro aggregates (P/PMN). These micro aggregates were visualized by light microscopy. Electron microscopy revealed no significant microvascular plugging. 1) A specifically blood-cell induced loss of myocardial pump function has been demonstrated after short-term ischemia. 2) CD41 (= GpIIbIIIa) on P is responsible for this cardiac reperfusion damage. 3) The effect is causally linked to the formation of micro aggregates between PMN and P, but seems attenuated in the presence of erythrocytes as compared to effects reported from experiments in which PMN and P were applied singly or co-perfused. 4) Intracoronary retention of PMN, M and platelet-leukocyte micro aggregates seems to be transient, as adherence was not confirmed by electron microscopy.

Relation between platelet microaggregates and ankle brachial index in patients with peripheral arterial disease

Introduction Peripheral arterial disease is one of the systemic atherosclerotic diseases, and patients with the disorder are classified in the high risk group of coronary artery disease. A lower ankle brachial index is a frequent finding in peripheral arterial disease. While platelet microaggregates are a significant predictor of adverse clinical outcome in coronary artery disease, the significance of platelet aggregability in peripheral arterial disease has not been elucidated. Materials and methods Small platelet aggregates measured using laser-light scattering and ankle brachial index were determined in 42 patients with both coronary artery disease and peripheral arterial disease (peripheral group), 56 patients with only coronary artery disease (coronary group) and 32 patients without both (control group). Results The level of small platelet aggregates was increased significantly in the peripheral group (4.3 × 10 4 [range 2.2 × 10 4 to 7.4 × 10 4]) compared with both the coronary (1.1 × 10 4 [range 0.3 × 10 4 to 5.0 × 10 4]) and control groups (0.5 × 10 4 [range 0.1 × 10 4 to 0.9 × 10 4]). There was a significant inverse correlation between log small platelet aggregates and ankle brachial index ( n = 130, r = − 0.422, p < 0.001). Multivariate logistic regression analysis revealed that a lower ankle brachial index (< 0.90) was an independent determinant of increased levels of small platelet aggregates. Conclusions Platelet aggregability was increased in patients with peripheral arterial disease with the degree of platelet aggregation being closely associated with ankle brachial index. It is possible that this change in platelet activity may be one mechanism to explain why a lower ankle brachial index is a predictor of poor prognosis in patients with peripheral arterial disease.

Upregulation of GP IIb/IIIa receptors during platelet activation: Influence on efficacy of receptor blockade

Introduction GP IIb/IIIa inhibitor doses high enough to inhibit platelet macroaggregation may not completely prevent formation of microaggregates. Platelets contain an internal pool of GP IIb/IIIa receptors which externalizes with activation and supports microaggregation. This study assesses microaggregation in the presence of the two GP IIb/IIIa inhibitors abciximab and tirofiban. Methods Citrated whole blood was preincubated with abciximab (5 μg/ml), tirofiban (50 ng/ml), or saline as control and activated with TRAP (5 μM) or ADP (2 μM) at 37 °C under constant stirring. Microaggregate formation and receptor expression were determined with flow cytometry. Results Within few seconds after TRAP-activation the platelet count dropped from 266,000/μl to 20,000/μl, the number of microaggregates increased from 3700/μl to 10,100/μl and the mean number of GP IIb/IIIa receptors increased from 53,000 to 65,000/platelet. With TRAP+abciximab the platelet count dropped from 259,000 to 113,000/μl, microaggregates increased from 2500 to 9300/μl, GP IIb/IIIa receptors from 56,000 to 77,000/platelet. Platelet microaggregate formation was reversible. With TRAP and tirofiban platelet count dropped to only 190,000/μl, there was no increase in platelet microaggregates, receptors increased to 66,000/platelet. Platelet activation with ADP gave similar results. Conclusions During the early phase of activation additional GP IIb/IIIa receptors externalize to the platelet surface. Abciximab does not block these new receptors sufficiently to prevent microaggregate formation. However, the number of unblocked receptors is not high enough to maintain a stable aggregate, microaggregation is reversible. With tirofiban there is no microaggregate formation, possibly because the inhibitor rapidly binds to newly externalized receptors.

Nafamostat Preserves Neutrophil Deformability and Reduces Microaggregate Formation During Simulated Extracorporeal Circulation

Initial sequestration of activated neutrophils and platelet microaggregates in capillaries are responsible for the inflammatory response associated with cardiopulmonary bypass. The study assesses the inhibitory effects of nafamostat mesilate on neutrophil and platelet activation, and on the neutrophil deformability change and microaggregate formation during simulated extracorporeal circulation. Fresh heparinized human blood was recirculated for 120 minutes in a membrane oxygenator and a roller pump with and without nafamostat (1.0 mg bolus plus 8.0 mg/h infusion; n = 10 for each group). Neutrophil and platelet counts and platelet aggregation were measured. CD11b, L-selectin, and cytoplasmic F-actin of neutrophils were measured by flow cytometry. The microchannel transit time of whole blood was measured as a marker of neutrophil deformability and microaggregate formation. Neutrophil elastase and complement C4d were measured using enzyme immunoassay. Nafamostat preserved platelet counts and inhibited platelet aggregation. Nafamostat significantly reduced neutrophil elastase release at 120 minutes of recirculation, and F-actin expression at 30 and 60 minutes. The drug did not modulate the changes of CD11b, L-selectin, or C4d. Whole blood filterability was significantly preserved by nafamostat at 30 and 120 minutes. Nafamostat preserves blood filterability during recirculation, possibly by suppression of F-actin expression and platelet activation. Nafamostat may reduce neutrophil sequestration and microaggregate formation in the microcirculation during cardiopulmonary bypass.

Characterization of a Heritable Bleeding Disorder in a Horse: Reduce PS-Flip Leads to Progressive Dysfunction in the Common Pathway of Platelet Coagulation.

A 3 year old Thoroughbred horse presented to the Veterinary Teaching Hospital with a three week history of bleeding. Clinical laboratory findings showed normal complete blood count including a normal platelet count. In addition the horse had normal prothrombin and partial thromboplastin times, as well as normal coagulation factors. These data suggested a defect in platelet function. The common pathway of the coagulation cascade occurs at the platelet surface when phosphatidylserine (PS) is flipped to the outer leaflet of the platelet membrane, providing binding sites for the prothrombinase complex that catalyzes the production of thrombin, which carries out the proteolytic activation of fibrinogen. We characterized the capacity of horse's platelets to carry out each of these steps. PS-flip in the patient's platelets was induced using several physiological agonists and found to be significantly reduced after stimulation with thrombin (0.1U/ml). The PS was detected by labeling platelets with FITC-annexin V, and measuring FITC intensity by flow cytometry. In comparison to control platelets, approximately 50% of the patient's platelets were positive for FITC-annexin V after stimulation with thrombin. The fluorescent intensity of the label on the patient's positive platelets was 40% that of controls. The reduced PS on the patient's platelets represents a potential loss of prothrombinase binding sites. Using a prothombinase assay based on the hydrolysis the chromogenic substrate S-2238, we determined the amount of thrombin produced by the patient's platelets over time. In response to thrombin (0.1U/ml), the patient's platelets produced thrombin at a rate of 0.023±0.002 NIH-U/min/1 X 108 platelets, which was significantly less than control platelets, 0.053±0.004 NIH-U/min/1 X 108 platelets. Neither resting platelets nor the thrombin used to stimulate them produced appreciable hydrolysis of S-2238. We correlated this reduced prothrombinase activity with the capacity of the patient's platelets bind FITC-fibrinogen. After stimulation with thrombin (0.1U/ml), control platelets form platelet microaggregates that are intensely labeled with FITC-fibrinogen and can be observed by flow cytometry. The patient's platelets do not form platelet microaggregates and the FITC-fibrinogen labeling is only 20% that of control platelets. This reduced FITC-fibrinogen labeling is not due to a defect in the function of the a2bb3a integrin, because the patient's platelets bind normal amounts of FITC-fibrinogen in response to ADP. In this regard, the affected horse is unlike patients with Glannzmann's thrombasthenia, who cannot bind fibrinogen. Instead, the dysfunction is more closely related to individuals with Scott syndrome, who cannot produce the PS surface that is required for assembly of the prothrombinase and ultimately activation of fibrinogen. Ongoing studies using the techniques described above demonstrate one affected and one normal progeny from the original patient.

The Cytoplasmic Tail of GPIbß Modulates Signalling Induced by GPIb/VWF Interactions under High Shear Conditions.

The platelet glycoprotein (GP) Ib/IX/V receptor complex controls crucial steps in haemostasis and thrombosis by mediating adhesion to von Willebrand factor (VWF). Nevertheless, the downstream consequences of VWF binding to GPIb remain unclear. The GPIb/IX/V complex consists of four polypeptides GPIbα, GPIbβ, GPIX and GPV. We have shown that a palmitylated (Pal-) peptide based on the sequence of the GPIbβ cytoplasmic domain between R151 and A161 (Pal-RRLRARARARA abbreviated to RARA) increases the velocity of platelets rolling on VWF, and inhibits VWF-dependent adhesion and aggregation.The peptide RARA is delivered into platelets and does not cause any reduction in the surface expression of GPIb-IX-V. We examined the effect of RARA and control peptides (Pal-RAAAARARARA and Pal-RRLRADADADA) on GPIb/IX/V-mediated tyrosine kinase signalling induced by VWF/GPIb interactions under shear conditions using a cone and plate viscometer. Whole platelet lysate phosphotyrosine immunoblots were characterised in the presence and absence of RARA and the control peptides before and after shearing at 120 dynes/cm2 for 5 mins. We then further investigated the effect of RARA on PI-3 kinase signalling, the tyrosine kinase Src and calcium influx under the conditions described above.The platelet permeable peptide RARA alone specifically caused protein tyrosine phosphorylation under resting conditions and caused platelet microaggregate formation with a 35% reduction in single platelets as assessed by flow cytometry. In addition RARA significantly inhibited shear-induced aggregation. When tyrosine phosphorylation was examined in platelets sheared for 5 minutes at 120 dynes/cm2, we observed RARA failed to inhibit tyrosine phosphorylation despite inhibiting shear-induced aggregation.RARA caused Src kinase activation as determined by site-specific phosphorylation of Y416 on Src. Furthermore the Src inhibitor PP1 inhibited RARA induced tyrosine phosphorylation. RARA caused a Src-dependent increase in intracellular [Ca2+] and inhibited any further increase in intracellular calcium induced by thrombin. Pretreatment of platelets with the PI-3 kinase inhibitor wortmannin demonstrated that tyrosine phosphorylation induced by RARA was not PI-3 kinase dependent.These results indicate that a platelet permeable peptide corresponding to specific residues of the cytoplasmic tail of GPIbβ directly stimulates Src, and suggest that the mechanism of its inhibitory effect on VWF-mediated platelet adhesion and aggregation involves the tyrosine kinase-mediated desensitisation of a GPIb/IX/V-triggered signalling pathway. This study demonstrates a novel role for the GPIbβ cytoplasmic tail in mediating Src-dependent shear induced signalling through GPIb/VWF.

P2Y12 receptors play a significant role in the development of platelet microaggregation in patients with diabetes.

Ninety-eight diabetic patients (type 2) were studied together with 24 healthy normotensive controls. Microaggregates (particle scale, <25 microm) of platelets were detected by a laser scattering system. Microaggregates in the control group showed a time-dependent reversible change; however, they existed continuously in 82 of 98 diabetic patients. When platelets of diabetics were stimulated by a shear stress alone without ADP, 74 also showed spontaneous and irreversible microaggregates even though they were not observed in all control subjects. In control subjects, microaggregates were inhibited by MRS2279 (a P2Y1 antagonist), but not AR-C69931MX (a P2Y12 antagonist). However, AR-C69931MX prevented irreversible microaggregates in diabetic patients. When either aspirin or ticlopidine was administered to diabetic patients with irreversible microaggregates, both drugs significantly decreased microaggregates induced by a low dose of ADP. Ticlopidine additionally reduced the microaggregates induced by shear stress alone. In conclusion, microaggregates of platelets via P2Y12 receptors could play a key role in the hypersensitivity of platelets in diabetic patients, and the measurement of microaggregation could be a useful marker to estimate of thrombogenesis. These findings present a possible new means for patients with diabetes to prevent ischemic events.