Molecular Imaging of Platelet-Endothelial Interactions and Endothelial von Willebrand Factor in Early and Mid-Stage Atherosclerosis.

BACKGROUND: Nonthrombotic platelet-endothelial interactions may contribute to atherosclerotic plaque development, although in vivo studies examining mechanism without platelet preactivation are lacking. Using in vivo molecular imaging at various stages of atherosclerosis, we quantified platelet-endothelial interactions and evaluated the contribution of major adhesion pathways. METHODS AND RESULTS: Mice deficient for the low-density lipoprotein receptor and Apobec-1 were studied as an age-dependent model of atherosclerosis at 10, 20, 30, and 40 weeks of age, which provided progressive increase in stage from early fatty streak (10 weeks) to large complex plaques without rupture (40 weeks). Platelet-targeted contrast ultrasound molecular imaging of the thoracic aorta performed with microbubbles targeted to GPIbα demonstrated selective signal enhancement as early as 10 weeks of age. This signal increased progressively with age (almost 8-fold increase from 10 to 40 weeks, analysis of variance P<0.001). Specificity for platelet targeting was confirmed by the reduction in platelet-targeted signal commensurate with the decrease in platelet count after immunodepletion with anti-GPIb or anti-CD41 antibody. Inhibition of P-selectin in 20 and 40 weeks atherosclerotic mice resulted in a small (15% to 30%) reduction in platelet signal. Molecular imaging with microbubbles targeted to the A1 domain of von Willebrand factor demonstrated selective signal enhancement at all time points, which did not significantly increase with age. Treatment of 20 and 40 week mice with recombinant ADAMTS13 eliminated platelet and von Willebrand factor molecular imaging signal. CONCLUSIONS: Platelet-endothelial interactions occur in early atherosclerosis. These interactions are in part caused by endothelial von Willebrand factor large multimers, which can be reversed with exogenous ADAMTS13.

Contrast-enhanced imaging of SPIO-labeled platelets using magnetomotive ultrasound.

The ability to image platelets in vivo can provide insight into blood clotting processes and coagulopathies, and aid in identifying sites of vascular endothelial damage related to trauma or cardiovascular disease. Toward this end, we have developed a magnetomotive ultrasound (MMUS) system that provides contrast-enhanced imaging of superparamagnetic iron oxide (SPIO) labeled platelets via magnetically-induced vibration. Platelets are a promising platform for functional imaging contrast because they readily take up SPIOs and are easily harvested from blood. Here we report a novel MMUS system that accommodates an arbitrarily thick sample while maintaining portability. We employed a frequency- and phase-locked motion detection algorithm based on bandpass filtering of the differential RF phase, which allows for the detection of sub-resolution vibration amplitudes on the order of several nanometers. We then demonstrated MMUS in homogenous tissue phantoms at SPIO concentrations as low as 0.09 mg ml(-1) Fe (p < 0.0001, n = 6, t-test). Finally, we showed that our system is capable of three-dimensional imaging of a 185 µL simulated clot containing SPIO-platelets. This highlights the potential utility for non-invasive imaging of platelet-rich clots, which would constitute a fundamental advance in technology for the study of hemostasis and detection of clinically relevant thrombi.

Microbubble mediated thrombus dissolution with diagnostic ultrasound for the treatment of chronic venous thrombi.

BACKGROUND: Central venous catheter (CVC) thrombi result in significant morbidity in children, and currently available treatments are associated with significant risk. We sought to investigate the therapeutic efficacy of microbubble (MB) enhanced sonothrombolysis for aged CVC associated thrombi in vivo. METHODS AND RESULTS: A model of chronic indwelling CVC in the low superior vena cava with thrombus in situ was established after feasibility and safety testing in 7 pigs; and subsequently applied for repeated, sonothrombolytic treatments in 9 pigs (total 24 treatments). Baseline intracardiac echocardiography (ICE, 10.5F, Siemens), fluoroscopy and saline flushing confirmed the absence of any pre-existing CVC thrombus. A thrombus was then allowed to form and age over 24 hours. The created thrombus was localized and measured by ICE, and transthoracic image guided high mechanical index (MI) two-dimensional US treatments (1.1-1.7 MI; iE33, Philips) applied intermittently whenever intravenously infused MBs (3% MRX-801; NuVox) were visualized near the thrombus (n = 10; Group A). Control pigs (n = 10; Group B) received US without MB. All treatments were randomized. Post-treatment thrombus area by ICE planimetry was compared with pre-treatment measurements. Thrombus area measurements before and after treatment were 0.22 and 0.10 cm(2) respectively in Group A; compared to 0.24 and 0.21 cm(2) in Group B (p  = 0.0003). Effectiveness of longer duration US and MB thrombolytic treatments were studied (n = 4), which suggested that near complete thrombus dissolution is possible. No pulmonary emboli, alterations in oxygen saturation, or hemodynamics occurred with either treatment. CONCLUSIONS: Guided high MI diagnostic US+systemic MB facilitates reduction of aged CVC associated thrombi in vivo. MB enhanced sonothrombolytic therapy may be a non-invasive safe alternative to thrombolytic agents in treating thrombotic CVC occlusions.

Inter-relationship between different platelet measures of 5-HT and their relationship to aggression in human subjects.

The objective of this study was to explore the inter-relationship of three platelet measures of serotonergic function (5-HT): 5-HT Transporter Binding, 5-HT-2 Receptor Binding and 5-HT Content and to explore their inter-relationship with measures of aggression and impulsivity. 58 male subjects with personality disorder were studied. Numbers of platelet 5-HT Transporter and 5-HT-2 Receptor sites were assessed by examining the Bmax of ³H-Paroxetine Binding and the Bmax of ¹²5I-LSD Binding to the blood platelet; 5-HT Content was assessed by measuring the amount of 5-HT in the platelet material. Life history of aggression was assessed by Life History of Aggression. Impulsivity was assessed by the Impulsivity Scale of the Eysenck Personality Questionnaire-II. Platelet 5-HT Transporter Binding correlated with both 5-HT-2 Receptor Binding and 5-HT Content; the latter two variables did not correlate with each other. Only Platelet 5-HT Transporter binding correlated significantly with LHA Aggression. These data suggest that while Platelet 5-HT Transporter binding correlates with both 5-HT-2 Receptor Binding and with 5-HT Content, that only 5-HT Transporter Binding represents a correlate of aggression in male personality disordered subjects.

The scintigraphic index spleen/liver at 30 minutes predicts the success of splenectomy in persistent and chronic primary immune thrombocytopenia.

UNLABELLED: Splenectomy is considered the second-line of treatment in patients with chronic primary immune thrombocytopenia (ITP) in whom glucocorticoids have failed. Some patients do not respond to splenectomy or they have postoperative complications. Based on our previous experience using kinetic and scintigraphic parameters, we did a retrospective study with the aim of comparing all these parameters as a means of predicting the success of splenectomy in persistent and chronic primary ITP. Forty-one consecutive patients with chronic primary ITP refractory to prednisone, who had been splenectomized, were included in the study. The response to splenectomy was assessed by evaluating bleeding and platelet counts before and at different times after surgery. A complete platelet kinetic study was performed before the splenectomy using autologous (111) In-labeled platelets. The scintigraphic parameters measured included different indices between spleen/heart, liver/hearth, and spleen/liver. Thirty-six patients gave a complete response after splenectomy and five patients did not respond. A statistically significant difference between both groups was found with initial platelet recovery and with some scintigraphic indices which also showed a variable prediction value for the success of splenectomy. Among these indices, the spleen/liver at 30 minutes demonstrated a predictive value with a 100% of sensitivity and a 100% of specificity. CONCLUSION: some platelet kinetic parameters and scintigraphic indices, in particular the spleen/liver at 30 minutes, were useful to predict the outcome of splenectomy in persistent and chronic primary ITP and, therefore, they should be taken into account when deciding whether or not to perform a splenectomy.

Radiolabeled fucoidan as a p-selectin targeting agent for in vivo imaging of platelet-rich thrombus and endothelial activation.

UNLABELLED: P-selectin expression is involved in the pathophysiology of biologically active arterial thrombus and endothelial activation after a transient ischemic event. Fucoidan is a polysaccharidic ligand of P-selectin, with a nanomolar affinity. In the present study, we propose a new approach of P-selectin molecular imaging based on radiolabeled fucoidan. METHODS: Two kinds of experimental models were selected to evaluate the ability of radiolabeled fucoidan to detect P-selectin expression: platelet-rich arterial thrombi (vegetations of infective endocarditis and arterial mural thrombus) and myocardial ischemia-reperfusion. These 2 settings were chosen because they were clinically relevant, and both were associated with an important overexpression of platelet and endothelial P-selectin, respectively. RESULTS: (99m)Tc-fucoidan SPECT was able to detect the presence of platelet-rich arterial thrombi in all animals, with a median target-to-background ratio of 5.2 in vegetations of endocarditis and 3.6 in mural aneurysmal thrombus, and to detect a persistent endothelial activation at 2 h after reperfusion. In this latter model, the magnitude of the signal was correlated with the extent of myocardium that underwent transient ischemia. The sensitivity of selectivity of the uptake and retention of (99m)Tc-fucoidan in both settings was excellent. CONCLUSION: This study supports (99m)Tc-fucoidan as a relevant imaging agent for in vivo detection of biologic activities associated with P-selectin overexpression, such as arterial thrombus and ischemic memory. Given the reported wide availability at a low cost, and its low toxicity, fucoidan seems to overcome some of the limitations of previous P-selectin-targeted imaging agents.

CT and MRI early vessel signs reflect clot composition in acute stroke.

BACKGROUND AND PURPOSE: The purpose of this study was to provide the first correlative study of the hyperdense middle cerebral artery sign (HMCAS) and gradient-echo MRI blooming artifact (BA) with pathology of retrieved thrombi in acute ischemic stroke. METHODS: Noncontrast CT and gradient-echo MRI studies before mechanical thrombectomy in 50 consecutive cases of acute middle cerebral artery ischemic stroke were reviewed blinded to clinical and pathology data. Occlusions retrieved by thrombectomy underwent histopathologic analysis, including automated quantitative and qualitative rating of proportion composed of red blood cells (RBCs), white blood cells, and fibrin on microscopy of sectioned thrombi. RESULTS: Among 50 patients, mean age was 66 years and 48% were female. Mean (SD) proportion was 61% (±21) fibrin, 34% (±21) RBCs, and 4% (±2) white blood cells. Of retrieved clots, 22 (44%) were fibrin-dominant, 13 (26%) RBC-dominant, and 15 (30%) mixed. HMCAS was identified in 10 of 20 middle cerebral artery stroke cases with CT with mean Hounsfield Unit density of 61 (±8 SD). BA occurred in 17 of 32 with gradient-echo MRI. HMCAS was more commonly seen with RBC-dominant and mixed than fibrin-dominant clots (100% versus 67% versus 20%, P=0.016). Mean percent RBC composition was higher in clots associated with HMCAS (47% versus 22%, P=0.016). BA was more common in RBC-dominant and mixed clots compared with fibrin-dominant clots (100% versus 63% versus 25%, P=0.002). Mean percent RBC was greater with BA (42% versus 23%, P=0.011). CONCLUSIONS: CT HMCAS and gradient-echo MRI BA reflect pathology of occlusive thrombus. RBC content determines appearance of HMCAS and BA, whereas absence of HMCAS or BA may indicate fibrin-predominant occlusive thrombi.

A(2A) adenosine receptor binding parameters in platelets from patients affected by pathological gambling.

BACKGROUND/AIMS: A structural and functional interaction between A(2A) adenosine receptors and D(2) dopamine receptors has been implicated in the pathophysiology of impulse control disorders. The aim of this study was to use platelet membranes to assess A(2A) adenosine receptor affinity and density in patients affected by pathological gambling (PG; which is classified as a specific impulse control disorder) with respect to those of control subjects. METHODS: Twelve drug-free PG patients and 12 age- and sex-matched healthy controls were enrolled in the study. PG was diagnosed according to the Structured Clinical Interview for DSM-IV - Patient Version 2.0 and the South Oaks Gambling Screen. A(2A) adenosine receptor binding parameters were evaluated using a [(3)H]ZM(241385) binding assay; affinity and density (B(max)) were determined by means of saturation binding studies with platelet membranes. RESULTS: The A(2A) adenosine receptor binding affinity was found to be significantly higher in patients affected by PG than in healthy subjects; in contrast, no significant differences in B(max) were observed between the 2 groups. CONCLUSIONS: The elevated A(2A) adenosine receptor binding affinity in platelets from PG patients with respect to control subjects demonstrates for the first time a change in adenosine receptor parameters, and it suggests the involvement of the adenosine system in this pathology. The previously demonstrated hyperactivity of the dopamine system in PG may modulate the A(2A) adenosine receptor, supporting a role for this receptor as a peripheral marker of dopamine dysfunction. Because it is not possible to directly measure the D(2) dopamine receptor in human platelets, these data are particularly relevant to the detection of dopamine dysfunction.

Elucidation of flow-mediated tumour cell-induced platelet aggregation using an ultrasound standing wave trap.

BACKGROUND AND PURPOSE: Tumour cells activate and aggregate platelets [tumour cell-induced platelet aggregation (TCIPA)] and this process plays an important role in the successful metastasis of cancer cells. To date, most studies on TCIPA have been conducted under no-flow conditions. In this study, we have investigated TCIPA in real time under flow conditions, using an ultrasound standing wave trap that allows formation and levitation of cancer cell clusters in suspension, thus mimicking the conditions generated by flowing blood. EXPERIMENTAL APPROACH: Using 59M adenocarcinoma and HT1080 fibrosarcoma cells and human platelets, cancer cell cluster-platelet aggregates were imaged in real time using epi-fluorescence microscopy (F-actin) and investigated in detail using confocal microscopy (matrix metalloproteinase-2-GPIIb/IIIa co-localization) and scanning electron and helium-ion microscopy (<1 nm resolution). The release of gelatinases from aggregates was studied using zymography. KEY RESULTS: We found that platelet activation and aggregation takes place on the surface of cancer cells (TCIPA), leading to time-dependent disruption of cancer cell clusters. Pharmacological modulation of TCIPA revealed that EDTA, prostacyclin, o-phenanthroline and apyrase significantly down-regulated TCIPA and, in turn, delayed cell cluster disruption, However, EGTA and aspirin were ineffective. Pharmacological inhibition of TCIPA correlated with the down-regulation of platelet activation as shown by flow-cytometry assay of platelet P-selectin. CONCLUSION AND IMPLICATIONS: Our results show for the first time, that during TCIPA, platelet activation disrupts cancer cell clusters and this can contribute to metastasis. Thus, selective targeting of platelet aggregate-cancer cell clusters may be an important strategy to control metastasis.

Autologous 111 In-labelled platelet sequestration studies in patients with primary immune thrombocytopenia (ITP) prior to splenectomy: a report from the United Kingdom ITP Registry.

While splenectomy is an effective therapy for primary immune thrombocytopenia (ITP), possible complications and observed non-complete response (CR) in one-third of patients demonstrate the need for further research into potential pre-surgical predictors of outcomes. Past investigations into platelet sequestration studies, a hypothesized predictive test, have adopted heterogeneous methods and varied widely with regard to power. By studying patients with primary ITP who underwent autologous (111) In-labelled platelet sequestration studies at Barts and The London NHS Trust between 1994 and 2008, we evaluated the effectiveness of sequestration site in predicting short, medium, and long-term CR (platelet count >100 × 10(9) /l) to splenectomy through multivariate (gender, age at splenectomy, and mean platelet lifespan) logistic regression modelling. In total, 256 patients with primary ITP underwent scans; 91 (35·5%) proceeded to splenectomy. Logistic regression revealed significant adjusted odds ratios for CR of 7·47 (95% confidence interval [CI], 1·89-29·43) at 1-3 months post-splenectomy, 4·85 (95% CI, 1·04-22·54) at 6-12 months post-splenectomy, and 5·39 (95% CI, 1·34-21·65) at last follow-up (median: 3·8 years [range: 0·5-13·1 years]) in patients with purely or predominantly splenic versus mixed or hepatic sequestration. These findings demonstrate the utility of autologous (111) In-labelled platelet sequestration studies as an adjunct predictive instrument prior to splenectomy.

An ultrasound contrast agent targeted to P-selectin detects activated platelets at supra-arterial shear flow conditions.

OBJECTIVES: To evaluate targeting of a microbubble contrast agent to platelets under high shear flow using the natural selectin ligand sialyl Lewis. MATERIALS AND METHODS: Biotinylated polyacrylamide Sialyl Lewis or biotinylated carbohydrate-free polymer (used as a control) were attached to biotinylated microbubbles via a streptavidin linker. Activated human platelets were isolated and attached to fibrinogen-coated culture dishes. Fibrinogen-coated dishes without platelets or platelet dishes blocked by an anti-P-selectin antibody served as negative control substrates. Dishes coated by recombinant P-selectin served as a positive control substrate. Microbubble adhesion was assessed by microscopy in an inverted parallel plate flow chamber, with wall shear stress values of 40, 30, 20, 10, and 5 dynes/cm2. The ratio of binding and passing microbubbles was defined as capture efficiency. RESULTS: There was no significant difference between the groups regarding the number of microbubbles in the fluid flow at each shear rate. Sialyl Lewis-targeted microbubbles were binding and slowly rolling on the surface of activated platelets and P-selectin-coated dishes at all the flow conditions including 40 dynes/cm2. Capture efficiency of targeted microbubbles to activated platelets and recombinant P-selectin decreased with increasing shear flow: at 5 dynes/cm2, capture efficiency was 16.11% on activated platelets versus 21.83% on P-selectin, and, at 40 dynes/cm2, adhesion efficiency was still 3.4% in both groups. There was neither significant adhesion of Sialyl Lewis-targeted microbubbles to control substrates, nor adhesion of control microbubbles to activated platelets or to recombinant P-selectin. CONCLUSIONS: Microbubble targeting using sialyl Lewis, a fast-binding ligand to P-selectin, is a promising strategy for the design of ultrasound contrast binding to activated platelets under high shear stress conditions.

Evidence-based mini-review: Is indium-labeled autologous platelet scanning predictive of response to splenectomy in patients with chronic immune thrombocytopenia?

Clinical scenario: An otherwise healthy 25-year-old woman returns to your office for management of chronic primary immune thrombocytopenia. She was diagnosed 6 months earlier and continues to require prednisone 15 mg daily and periodic infusions of intravenous immunoglobulin to maintain a hemostatic platelet count. You discuss second-line treatment options, including splenectomy. The patient asks if there are any means by which to predict likelihood of response to splenectomy. You have heard about the use of indium-labeled autologous platelet scanning for this purpose and wonder what the evidence shows.

An experimental study of the use of ultrasound to facilitate the loading of trehalose into platelets.

Trehalose is widely used as a freeze-drying protectant in biomaterial preservation. For this purpose, trehalose has to be loaded into the cells but this is difficult and many methods have been tried. The application of ultrasound can temporarily permeabilize cell membranes, which offers a non-chemical, non-viral, and non-invasive method of cellular drug delivery. Ultrasound is employed here to enhance the loading of trehalose into human platelets. Two frequencies were used, 25 kHz and 800 kHz. The estimated intensity of ultrasound in the sample was varied from 0 to 1.5 W/cm(2). The trehalose concentration in the platelets was 11.27+/-2.53 mmol/L when Wolkers et al.'s method was used without ultrasound. The application of 0.8 W/cm(2), 800 kHz ultrasound for 1h increased the concentration of trehalose loaded by 54%. The application of 0.8 W/cm(2), 25 kHz ultrasound for 30 min increased the trehalose concentration that was loaded by 172%. The number and mean volume of the platelets following ultrasonic radiation in these two cases remained normal as compared with fresh untreated platelets. Morphological examination of the radiated platelets showed slight changes. Although further work is needed, ultrasound has been shown to be efficient for the loading of trehalose into platelets.

Mean platelet volume, an indicator of platelet activation, is increased in patients with mitral stenosis and sinus rhythm.

Systemic thromboembolism is a serious major complication in patients with mitral stenosis. However, the pathogenesis of thromboembolism in mitral stenosis is not fully understood. Previous studies have demonstrated that platelet activation occurs in serum of patients with rheumatic mitral stenosis (MS). The aim of this study was to assess the mean platelet volume (MPV), an indicator of platelet activation, in patients with MS. The study group consisted of 36 patients with MS who were in sinus rhythm. An age and gender matched control group was composed of 30 healthy volunteers. We measured serum MPV values in patients and control subjects. MPV was significantly higher among MS patients with sinus rhythm (SR) when compared with the control group (9.2+/-1.4 vs 8.1+/-0.9 fl respectively; p<0.001). We have shown that MPV was significantly elevated in patients with MS who were in SR compared to control subjects.

Microembolic signals on transcranial Doppler ultrasound are correlated with platelet activation markers, but not with platelet-leukocyte associates: a study in patients with acute stroke and in patients with asymptomatic carotid stenosis.

BACKGROUND: The in vivo correlates of microembolic signals (MES) are still unknown. Platelet-associates (PA) with monocytes or granulocytes or platelet aggregates only may represent these correlates. METHODS: Thirty patients with asymptomatic carotid stenosis >50% and 16 patients with acute (<4 days) atherothrombotic stroke were investigated. PA, P-selectin and thrombospondin expressions on platelets were assessed by flow cytometry. Soluble P-selectin (sPS) levels were assessed. MES detections were performed by transcranial Doppler sonography for 1 hour. PA, P-selectin and thrombospondin expressions on platelets and sPS levels were compared between MES-positive (MES+) and MES-negative (MES-) patients. RESULTS: Eight patients (27%) with asymptomatic carotid stenosis had 1-26 MES/h. Degree of stenosis was 78 +/- 10% in MES- and 88 +/- 8% in the MES+ (p=0.01). There were no differences in percentages of PA. P-selectin and thrombospondin surface expression was lower in MES+, but this was not significant. sPS levels were higher in MES+ (122 +/- 27 ng/ml versus 80 +/- 25 ng/ml in MES-, p=0.01). Seven (44%) patients with stroke had 1-39 MES/h. There were no differences in percentages of PA. MES+ had higher sPS levels (178 +/- 43 versus 121 +/- 44 ng/ml, p=0.02) and less P-selectin surface expression than MES- (9.0 +/- 3.4 versus 4.5 +/- 1.6%, p=0.004). CONCLUSION: High levels of sPS in MES+ and lower expression of platelet activation markers on platelets' surface suggest shedding of activation markers from the platelets' surface and thus enhanced activation of platelets of MES+ compared with MES-. PA are probably not the clinical correlates of MES, but platelets seem to be the main cellular element of solid cerebral microemboli.

Identifying vulnerable carotid plaques by noninvasive imaging.

Stroke results in considerable morbidity and mortality. Prevention is therefore of particular importance. On the basis of large clinical trials, carotid endarterectomy (CEA) is performed in selected patient groups to prevent stroke. Patient symptomatology and degree of carotid stenosis are the main clinical grounds to perform CEA. However, many individual patients undergo surgery with its attendant risks without taking advantage of it, whereas in others CEA is probably incorrectly withheld. There is therefore an urgent need for new adjuncts to identify high-risk subgroups of patients who particularly benefit from potentially hazardous interventions. Multiple noninvasive imaging modalities have shown their potential to differentiate high-risk, vulnerable carotid plaques from stable plaques. The ultimate goal is to implement one or a combination of these imaging modalities in daily clinical practice. This review gives an up-to-date overview of the clinical potential of these imaging modalities in identifying patients with carotid atherosclerosis who are at high risk for developing stroke. Advantages and limitations of each imaging technique are outlined. Additionally, recommendations for future research are presented.

Platelet binding and biodistribution of [99mTc]rBitistatin in animal species and humans.

INTRODUCTION: 99mTc recombinant bitistatin (rBitistatin) is a radioligand for alphaIIbbeta3 (glycoproteins IIb/IIIa) receptor on platelets and is being developed as a diagnostic radiopharmaceutical for in vivo imaging of acute thrombi and emboli. Prior to the first administration of [99mTc]rBitistatin to human subjects, its biodistribution and effects on platelets were evaluated in animals. This paper reports findings in animal studies in comparison with initial findings in normal human subjects. METHODS: [99mTc]rBitistatin was administered to mice, guinea pigs and dogs to assess time-dependent organ distribution, urinary excretion and blood disappearance rates. Blood samples were analyzed to determine radioligand binding to circulating platelets and the extent of plasma protein binding. The effect of [99mTc]rBitistatin on circulating platelet count was determined. These factors were also determined in normal human subjects who received [99mTc]rBitistatin as part of a Phase I clinical trial. RESULTS: The main organs that accumulated [99mTc]rBitistatin were kidneys, liver and spleen in all animal species and humans. The main organs seen on human images were the kidneys and spleen. Liver uptake was fainter, and soft-tissue background was low. [99mTc]rBitistatin bound to circulating platelets in blood, with a higher percentage of binding to platelets in guinea pigs and dogs compared to that in humans. Plasma protein binding was low and of little consequence in view of platelet binding. The main route of excretion was through the urine. [99mTc]rBitistatin did not affect platelet counts in humans or dogs. CONCLUSIONS: [99mTc]rBitistatin, when administered at low doses for imaging, has no adverse effects on platelets and has the qualitative biodistribution predicted by animal studies. [99mTc]rBitistatin was found to bind to circulating platelets in humans, suggesting that it will be able to bind to activated platelets in vivo in patients with acute thrombi.