Total Thrombus Area Research Articles

Abstract 136: Protease-activating Receptor-4 Antagonism In Coronary Artery Disease

Background: BMS-986141 is a novel potent highly selective antagonist of protease-activated receptor type 4 (PAR4). PAR4 antagonism has been demonstrated to reduce thrombus formation in isolation and in combination with factor Xa inhibition in high shear conditions in healthy people. Aim: To determine whether PAR4 antagonism had additive antithrombotic effects in patients with coronary artery disease who were receiving antiplatelet therapy. Methods: Forty-five patients with stable coronary heart disease and ten healthy volunteers completed a phase 2a open-label four-arm single-center study. Patients were allocated to one of three treatment arms for seven days: (i) ticagrelor (90 mg twice daily), (ii) aspirin (75 mg once daily) or (iii) the combination of ticagrelor and aspirin. Agonist-induced platelet aggregation, platelet activation, and ex vivo thrombus formation were measured before, 2 and 24 hours after a single oral 4-mg dose of BMS-986141 in all participants. Results: BMS-986141 demonstrated highly selective inhibition of PAR4-agonist peptide (AP) induced platelet aggregation, p-selectin expression, and platelet-monocyte aggregate expression (p≤0.001 for all) which were unaffected by concomitant antiplatelet therapies. PAR4 antagonism reduced ex vivo thrombus area in high shear conditions in healthy volunteers (-21%, p=0.001) and in patients receiving ticagrelor alone (-28%, p=0.001), aspirin alone (-23%, p=0.018), or both in combination (-24%, p≤0.001). Plasma concentration of BMS-986141 correlated with PAR4-AP-induced platelet responses (p≤0.001 for all) and total thrombus area under high shear stress conditions (p≤0.01 for all). Conclusions: PAR4 antagonism has additive antithrombotic effects when used in addition to ticagrelor, aspirin or their combination, in patients with stable coronary artery disease.

PAR4 Antagonism in Patients With Coronary Artery Disease Receiving Antiplatelet Therapies.

BMS-986141 is a novel potent highly selective antagonist of PAR (protease-activated receptor) type 4. PAR4 antagonism has been demonstrated to reduce thrombus formation in isolation and in combination with factor Xa inhibition in high shear conditions in healthy people. We sought to determine whether PAR4 antagonism had additive antithrombotic effects in patients with coronary artery disease who were receiving antiplatelet therapy. Forty-five patients with stable coronary heart disease and 10 healthy volunteers completed a phase 2a open-label 4-arm single-center study. Patients were allocated to 1 of 3 treatment arms for 7 days: (1) ticagrelor (90 mg BID), (2) aspirin (75 mg QD), or (3) the combination of ticagrelor and aspirin. Agonist-induced platelet aggregation, platelet activation, and ex vivo thrombus formation were measured before and 2 and 24 hours after a single oral 4-mg dose of BMS-986141 on the first study visit day in all participants. BMS-986141 demonstrated highly selective inhibition of PAR4-AP (agonist peptide)-induced platelet aggregation, P-selectin expression, and platelet-monocyte aggregate expression (P≤0.001 for all), which were unaffected by concomitant antiplatelet therapies. PAR4 antagonism reduced ex vivo thrombus area in high shear conditions in healthy volunteers (-21%; P=0.001) and in patients receiving ticagrelor alone (-28%; P=0.001), aspirin alone (-23%; P=0.018), or both in combination (-24%; P≤0.001). Plasma concentration of BMS-986141 correlated with PAR4-AP-induced platelet responses (P≤0.001 for all) and total thrombus area under high shear stress conditions (P≤0.01 for all). PAR4 antagonism has additive antithrombotic effects when used in addition to ticagrelor, aspirin, or their combination, in patients with stable coronary heart disease. URL: https://www.clinicaltrials.gov; Unique identifier: NCT05093790.

Antithrombotic Effects of Combined PAR (Protease-Activated Receptor)-4 Antagonism and Factor Xa Inhibition.

PAR (protease-activated receptor)-4 antagonism has antiplatelet effects under conditions of high shear stress. We aimed to establish whether PAR4 antagonism had additive antithrombotic activity in the presence of factor Xa inhibition in an ex vivo model of acute arterial injury. Approach and Results: Fifteen healthy volunteers (29±6 years, 7 women) completed a phase zero double-blind randomized controlled crossover trial. Ex vivo platelet activation, platelet aggregation, and thrombus formation were measured following blood perfusion of low shear and high shear stress chambers. Upstream of the chambers, extracorporeal blood was admixed with (1) vehicle, (2) low-dose apixaban (20 ng/mL), (3) high-dose apixaban (80 ng/mL), (4) BMS-986141 (400 ng/mL), (5) BMS-968141 and low-dose apixaban, or (6) BMS-968141 and high-dose apixaban in 6 sequential studies performed in random order. Compared with vehicle, BMS-986141 demonstrated selective inhibition of PAR4-AP (agonist peptide)-stimulated platelet aggregation, platelet-monocyte aggregates, and P-selectin expression (P≤0.01 for all). Total thrombus area was reduced under both low shear and high shear stress conditions for all drug infusions (P<0.0001 for all versus vehicle). BMS-968141 reduced total (≤44.4%) and platelet-rich (≤39.3%) thrombus area, whereas apixaban reduced total (≤42.9%) and fibrin-rich (≤31.6%) thrombus area. Combination of BMS-986141 with apixaban caused a further modest reduction in total thrombus area (9.6%-12.4%), especially under conditions of high shear stress (P≤0.027). In the presence of factor Xa inhibition, PAR4 antagonism with BMS-986141 further reduces thrombus formation, especially under conditions of high shear stress. This suggests the potential for additive efficacy of combination PAR4 antagonism and factor Xa inhibition in the prevention of atherothrombotic events.

SLC44A2 deficient mice have a reduced response in stenosis but not in hypercoagulability driven venous thrombosis

BackgroundGenome wide association studies (GWAS) identified SLC44A2 as a novel susceptibility gene for venous thrombosis (VT) and previous work established that SLC44A2 contributed to clot formation upon vascular injury. ObjectiveTo further investigate the role of SLC44A2 in VT by utilizing SLC44A2 deficient mice (Slc44a2−/−) in two representative disease models. MethodsMice were included in a hypercoagulability model driven by siRNA‐mediated hepatic gene silencing of anticoagulants Serpinc1 (antithrombin) and Proc (protein C) and a flow restriction (stenosis) model induced by partial ligation of the inferior vena cava. ResultsIn the hypercoagulability model, no effect in onset was observed in Slc44a2−/− animals; however, a drop in plasma fibrinogen and von Willebrand factor coinciding with an increase in blood neutrophils was recorded. In the neutrophil dependent stenosis model after 48 hours, Slc44a2−/− mice had significantly smaller thrombi both in length and weight with less platelet accumulation as a percentage of the total thrombus area. During the initiation of thrombosis at 6 hours post‐stenosis, Slc44a2−/− mice also had smaller thrombi both in length and weight, with circulating platelets remaining elevated in Slc44a2−/− animals. Platelet activation and aggregation under both static‐ and venous and arterial shear conditions were normal for blood from Slc44a2−/− mice. ConclusionsThese studies corroborate the original GWAS findings and establish a contributing role for SLC44A2 during the initiation of VT, with indications that this may be related to platelet‐neutrophil interaction. The precise mechanism however remains elusive and warrants further investigation.

Diagnostic utility and clinical implication of late gadolinium enhancement cardiac magnetic resonance for detection of catheter associated right atrial thrombus

PurposeTo use delayed enhancement cardiac magnetic resonance (DE-CMR) as a reference standard to evaluate the prevalence and predictors of right atrial (RA) thrombus. MethodsIn this retrospective study, 130 cancer patients with central venous catheters undergoing CMR from August 2012–January 2018 were included. CMR (cine-CMR and DE-CMR) and echocardiography were interpreted for RA thrombus blinded to other imaging results and clinical data. RA thrombus properties including the number of discrete masses, size, total thrombus area, and perimeter were also assessed. Cine-CMR was also used to quantify cardiac structure and function as markers of RA thrombus. Student's t-test was used to assess continuous variables; chi-square or Fisher's exact test were used to assess categorical variables. Results31/130 (24%) patients had RA thrombus on DE-CMR. Echocardiography (attained in 64% of the study population) demonstrated moderate sensitivity and specificity (75%, 90% respectively) in relation to DE-CMR; cine-CMR performance was higher (sensitivity 90%, specificity 98%). Patients with and without RA thrombus had similar right-sided structure/function and cancer diagnosis. Catheter depth approached significance in patients with RA thrombus (p = 0.05). 13% of patients with RA thrombus had concomitant pulmonary embolism within 60 days of CMR vs. 2% of patients without RA thrombus (p = 0.03). Embolic events were independent of RA thrombus size (p = 0.66). ConclusionMorphologic imaging by cine-CMR and echocardiography provide limited diagnostic utility for RA thrombus as established by DE-CMR tissue characterization. Catheter-associated RA thrombus occurs independently of right-sided structure or function and is associated with clinical embolic events and catheter depth.

Exosite 1 thrombin inhibition with JNJ-64179375 inhibits thrombus formation in a human translational model of thrombosis.

AimsJNJ-64179375 (hereafter JNJ-9375) is a first-in-class, highly specific, large molecule, exosite 1 thrombin inhibitor. In preclinical studies, JNJ-9375 demonstrated robust antithrombotic protection with a wider therapeutic index when compared to apixaban. The purpose of the present study was to examine for the first time the antiplatelet, anticoagulant and antithrombotic effects of JNJ-9375 in a translational model of ex vivo human thrombosis.Methods and resultsFifteen healthy volunteers participated in a double-blind randomized crossover study of JNJ-9375 (2.5, 25, and 250 μg/mL), bivalirudin (6 μg/mL; positive control), and matched placebo. Coagulation, platelet activation, and thrombus formation were determined using coagulation assays, flow cytometry, and an ex vivo perfusion chamber, respectively.JNJ-9375 caused concentration-dependent prolongation of all measures of blood coagulation (prothrombin time, activated partial thromboplastin time, and thrombin time; P < 0.001 for all) and agonist selective inhibition of thrombin (0.1 U/mL) stimulated platelet p-selectin expression (P < 0.001) and platelet-monocyte aggregates (P = 0.002). Compared to placebo, JNJ-9375 (250 μg/mL) reduced mean total thrombus area by 41.1% (95% confidence intervals 22.3 to 55.3%; P < 0.001) at low shear and 32.3% (4.9 to 51.8%; P = 0.025) at high shear. Under both shear conditions, there was a dose-dependent decrease in fibrin-rich thrombus (P < 0.001 for both) but not platelet-rich thrombus (P = ns for both).ConclusionExosite 1 inhibition with JNJ-9375 caused prolongation of blood coagulation, selective inhibition of thrombin-mediated platelet activation, and reductions in ex vivo thrombosis driven by a decrease in fibrin-rich thrombus formation. JNJ-9375 represents a novel class of anticoagulant with potential therapeutic applications.

Neutrophil extracellular traps in ischemic stroke thrombi.

Neutrophil extracellular traps (NETs) have been shown to promote thrombus formation. Little is known about the exact composition of thrombi that cause ischemic stroke. In particular, no information is yet available on the presence of NETs in cerebral occlusions. Such information is, however, essential to improve current thrombolytic therapy with tissue plasminogen activator (t-PA). This study aimed at investigating the presence of neutrophils and more specifically NETs in ischemic stroke thrombi. Sixty-eight thrombi retrieved from ischemic stroke patients undergoing endovascular treatment were characterized by immunostaining using neutrophil markers (CD66b and neutrophil elastase) and NET markers (citrullinated histone H3 [H3Cit] and extracellular DNA). Neutrophils and NETs were quantified. In addition, extracellular DNA was targeted by performing ex vivo lysis of retrieved thrombi with DNase 1 and t-PA. Neutrophils were detected extensively throughout all thrombi. H3Cit, a hallmark of NETs, was observed in almost all thrombi. H3Cit-positive area varied up to 13.45% of total thrombus area. Colocalization of H3Cit with extracellular DNA released from neutrophils confirmed the specific presence of NETs. H3Cit was more abundant in thrombi of cardioembolic origin compared to other etiologies. Older thrombi contained significantly more neutrophils and H3Cit compared to fresh thrombi. Interestingly, ex vivo lysis of patient thrombi was more successful when adding DNase 1 to standard t-PA. Neutrophils and NETs form important constituents of cerebral thrombi. Targeting of NETs with DNase 1 might have prothrombolytic potential in treatment of acute ischemic stroke. Ann Neurol 2017;82:223-232.

The Uremic Toxin Indoxyl Sulfate Accelerates Thrombotic Response after Vascular Injury in Animal Models.

Chronic kidney disease (CKD) patients are at high risk for thrombotic events. Indoxyl sulfate (IS) is one of the most potent uremic toxins that accumulates during CKD. Even though IS is associated with an increased risk for cardiovascular disease, its impact on thrombotic events still remains not fully understood. The purpose of the study was to evaluate the direct effect of IS on thrombotic process. We examined the impact of acute exposure to IS on thrombus development induced by electric current in Wistar rats, intravital thrombus formation after laser-induced injury in the mice endothelium, coagulation profile, clot formation dynamics, platelet aggregations, and erythrocyte osmotic resistance. IS doses: 10, 30 and 100 mg/kg body weight (b.w.) increased weight of thrombus induced by electric current in dose-dependent manner (p < 0.001). Furthermore, two highest IS doses increased laser-induced thrombus formation observed via confocal system (increase in fluorescence intensity and total thrombus area (p < 0.01)). Only the highest IS dose decreased clotting time (p < 0.01) and increased maximum clot firmness (p < 0.05). IS did not affect blood morphology parameters and erythrocyte osmotic resistance, but augmented collagen-induced aggregation. Obtained data indicate that IS creates prothrombotic state and contributes to more stable thrombus formation. Thus, we concluded that IS may be one of crucial uremic factors promoting thrombotic events in CKD patients.

113 Blood thrombogenicity is inversely related to coronary lesion severity in patients with non ST-elevation acute coronary syndrome and type 2 diabetes mellitus

In patients with type 2 diabetes mellitus (T2DM), non-ST elevation–acute coronary syndrome (NSTE-ACS) occurs frequently secondary to non-obstructive coronary lesions than those without T2DM. Pathophysiological events leading to acute myocardial...

Thrombosomes Show Dose-Dependent Increase in Thrombus Formation in a Model of Deep Arterial Injury

Abstract 2319 IntroductionThrombosomes are novel lyophilized human platelet derivatives that retain a number of key platelet structural and functional properties. Building on preliminary in vitro studies, we here sought to investigate whether Thrombosomes, suspended in platelet free plasma (PFP), would enhance and be incorporated in thrombus generated under flow conditions within a validated and well-characterised model of deep arterial injury. MethodsPFP was obtained by centrifuging citrated whole blood from six healthy non-smoking volunteers and filtering with a 0.22μm filter. Thrombosomes were suspended in 60 mL of PFP to generate final concentrations of 20 and 200 × 106Thrombosomes /mL. Immediately prior to use, 1.2 mL of 1M CaCl2 was added to permit fibrin generation. Thrombus formation was assessed using the Badimon Chamber at low (212 s−1) and high (1690 s−1) shear rates with porcine aortic tunica media as the thrombogenic substrate. Total thrombus area and platelet-rich area were measured histomorphometrically using conventional and immunohistochemical staining respectively. Fluorescent labeled Thrombosomes were added to the extracorporeal circuit in the Badimon chamber to study the ex vivo thrombus generation in the whole blood. Electron microscopy of Thrombosomes and platelets was undertaken. ResultsThrombosomes contributed towards thrombus formation in whole human blood as evidenced by incorporation of fluorescent-labeled Thrombosomes into the thrombus. Immunohistochemical staining of the glycoprotein IIb/IIIa receptor confirmed incorporation of Thrombosomes into the thrombus. In the high shear chamber, mean thrombus area increased in a dose-dependent manner following the addition of Thrombosomes (704 μm2 [95% CI, 224 – 1184 μm2], 1511 μm2 [95% CI, 687 – 2336 μm2] and 2378 μm2 [95% CI, 1567 – 3189 μm2] for PFP and Thrombosomes at concentrations of 20 and 200 × 106/mL respectively [P= 0.003]). In the low shear chamber, total thrombus area for the PFP was 4962 μm2 (95% CI, 2489 – 7434 μm2). The addition of Thrombosomes at concentrations of 20 and 200 × 106/mL led to a numerical increase in mean thrombus area to 6170 μm2 (95% CI, 3944 – 8397 μm2) and 7504 μm2 (95% CI, 3864 – 11144 μm2) respectively, although this was not statistically significant (P= 0.2969). ConclusionsThrombosomes suspended in PFP and exposed to injured arterial tissue under physiologically relevant flow conditions are incorporated into thrombus and enhance thrombus formation in a dose dependent manner. These findings act as impetus to undertake clinical studies of this rehydrated, lyophilized infusible hemostatic platelet product. [Display omitted] [Display omitted] [Display omitted] Disclosures:Fitzpatrick:Cellphire Inc.: Employment, Equity Ownership. Feuerstein:Cellphire: Consultancy. Newby:Cellphire: Research Funding.

Activated inflammatory cells participate in thrombus size through tissue factor and plasminogen activator inhibitor-1 in acute coronary syndrome: Immunohistochemical analysis

Introduction Recent studies have suggested that circulating inflammatory cells augment the growth of thrombus in acute coronary syndrome (ACS). We therefore immunohistochemically analyzed thrombi in aspirates obtained from patients immediately after the onset of ACS. Materials and Methods Two hundred twenty samples were studied. Total thrombus area, white thrombus area, and red thrombus area were measured. As antibodies in immunohistochemical staining, myeloperoxidase (MPO), CD66b, CD68, p-selectin, tissue factor (TF) and PAI-1were employed respectively. Results The ratios of areas of red and white thrombi correlated with whole sample areas of enlarged thrombi (r = 0.48, p < 0.001). The immunohistochemical findings revealed granulocytes and macrophages aggregated around p-selectin-positive platelets that shared the boundary between white and red thrombi, a region where MPO and CD66b expression was abundant in neutrophils. The ratios (%) of MPO- and CD66b-positive cells significantly correlated with whole sample areas (r = 0.50; p < 0.001 and r = 0.49; p < 0.001, respectively). Neutrophils and macrophages within thrombi were positive for TF and PAI-1. Along the boundary between red and white thrombi, TF and PAI-1 positivity coincided with MPO-, CD66b- and CD68-positive cells. The ratios of cells positive for both TF and PAI-1 in this area significantly correlated with the whole sample area (r = 0.43, p < 0.001 and r = 0.60, p < 0.001, respectively). Conclusions These results suggested that enhanced activation of peripheral neutrophils together with increased TF and PAI-1 expression might comprise a considerable portion of thrombus enlargement.

Characterisation and reproducibility of a human ex vivo model of thrombosis

Background The Badimon chamber is a clinical ex vivo model of thrombosis that mimics flow conditions within the coronary circulation of man. The aims of this study were to characterise thrombus formation in the chamber and evaluate its reproducibility. Methods Using blood from 24 healthy human volunteers, thrombus formation was assessed at low and high shear rates with porcine aortic tunica media as the thrombogenic substrate. Thrombus area was measured histomorphometrically. Reproducibility was assessed by paired measurements made both within and between days. Platelet activation was assessed before and at selected points within the extracorporeal circuit using flow cytometry, and fibrin content and distribution within the thrombus were assessed by immunohistochemistry. Results Total thrombus area was highly reproducible within and between days in the low shear ([mean thrombus area, mean difference ± SEM] 8,018 μm 2, 58 ± 204 μm 2 and 8,177 μm 2, -154 ± 168 μm 2 respectively) and high shear chambers (11,802 μm 2, -52 ± 175 μm 2 and 11,877 μm 2, 220 ± 181 μm 2 respectively). Total thrombus area was greater in the high compared to the low shear chamber (11,970 ± 285 μm 2 versus 7,892 ± 298 μm 2; P < 0.0001). Transit through the extracorporeal circuit did not result in platelet activation which was only detected after blood passed across the perfusion chambers (P = 0.02 for platelet-monocyte aggregate formation and P = 0.05 for P-selectin expression). Thrombus in the low shear chamber contained a greater proportion of fibrin (25.0 ± 6.0% versus 8.3 ± 1.6%, P < 0.001). Conclusions The Badimon chamber provides a highly reproducible technique for the assessment of ex vivo platelet-rich thrombus formation in man.

P-selectin antagonism reduces thrombus formation in humans.

Interaction of P-selectin with its glycoprotein ligand (P-selectin glycoprotein ligand type 1) mediates inflammatory processes that may also include vascular thrombosis. Platelet P-selectin expression is increased in patients with coronary heart disease, and its antagonism represents a potential future therapeutic target for the prevention and treatment of atherothrombosis. To investigate the effects of the novel small molecule P-selectin antagonist PSI-697 on thrombus formation in humans. In a double-blind randomized crossover study, thrombus formation was measured in 12 healthy volunteers, using the Badimon ex vivo perfusion chamber under conditions of low and high shear stress. Saline placebo, low-dose (2 m) and high-dose (20 m) PSI-697 and the glycoprotein IIb-IIIa receptor antagonist tirofiban (50 ng mL(-1)) were administered into the extracorporeal circuit prior to the perfusion chamber. As compared with saline placebo, blockade of platelet glycoprotein IIb-IIIa receptor with tirofiban produced 28% and 56% reductions in thrombus formation in the low-shear and high-shear chambers, respectively. PSI-697 caused a dose-dependent, but more modest, reduction in thrombus formation. Low-dose PSI-796 (2 m) reduced total thrombus area by 14% (P = 0.04) and 30% (P = 0.0002) in the low-shear and high-shear chambers, respectively. At the high dose (20 m), PSI-697 reduced total thrombus area by 18% (P = 0.0094) and 41% (P = 0.0008) in the low-shear and high-shear chambers, respectively. P-selectin antagonism with PSI-697 reduces ex vivo thrombus formation in humans. These findings provide further evidence that P-selectin antagonism may be a potential target for the prevention and treatment of cardiovascular disease.

In Vivo Imaging of Large-Vessel Thrombosis

In vivo thrombosis imaging systems have been developed that show localized smallscale and short-acting thrombotic events in arterioles and venules; however, these microvascular models may not accurately simulate thrombogenesis in large vessels. A new quantitative imaging system was developed for evaluation of electrolytic-injury models of thrombus induction in large vessels of mice (carotid arteries and femoral veins). Laser-induced fluorophore emissions of membrane-labeled platelets and labeled fibrin-specific monoclonal antibodies were captured with time-lapse video recordings. Platelets were seen to accrue slowly after thrombus induction in both arteries and veins, peaking at approximately 15–45 minutes. In arteries, platelets accumulated more rapidly and to a greater extent than fibrin. Surprisingly, platelet accumulation in veins was substantial, occurring diffusely throughout the thrombus. Arterial thrombus dissolution occurred most often via large embolic events, sometimes occurring cyclically every 5–20 minutes by regrowth and re-embolization. In contrast, the rate of thrombus dissolution in veins was more gradual and appeared as micro-embolic events. Standard heparin anticoagulation inhibited venous thrombus growth by reducing fibrin development by 75% without affecting platelet accumulation. In contrast, blocking platelet aggregation at the αIIb/β3 receptor (with RGD-based mimetics) resulted in a 43% reduction in platelets and a surprising 5-fold increase in fibrin accumulation, suggesting the possibility of temporal competition between platelets and fibrin under venous conditions of thrombus formation. In established thrombi, newly activated platelets, identified by exposure of the active site of the αIIb/β3 receptor (using JON/A, a fluorophore-labeled antibody specific to activated αIIb/β3), were seen to roll in small aggregates across the outer margin of the thrombus mass in both arteries and veins for over 30 minutes, indicating continual dynamic platelet aggregation in both vessel types. Fibrin accrual was most intense at the margins of acute thrombotic stimuli and co-localized with fluorophore-labeled anti-Factor Xa and with Factor Va/VIIIa (identified using labeled activated protein C), which suggests that fibrin polymerization predominates adjacent to sites of these assembled coagulation complexes. In summary, the time-lapse nature of this video imaging system provides a unique and quantifiable method for determining how coagulation and platelet aggregation develop and are modulated in vivo within the same thrombus. Its use for the study of large vessel thrombosis often corroborated long-held tenets of platelet activity and coagulation, while revealing several novel findings:platelets are major contributors to venous thrombosis;arteries and veins have profound differences in thrombus dissolution/embolization patterns;inhibiting platelet aggregation may augment fibrin clot formation;platelet aggregation is a dynamic process in both arteries and veins, continually in flux on the thrombus surface for at least 30 minutes; andfibrin forms local to sites of coagulation complex assembly. [Display omitted] Graph illustrating single traces for representative electrolytically-induced, free-radical-mediated thrombi in mouse carotid arteries (EAT) and femoral veins (EVT) over 60 minutes, for anti-fibrin (FBN) and platelet (Plt) labels. The y-axis displays arbitrary units for the product of total thrombus area and average fluorophore intensity within the thrombus.

Antithrombotic effects of ximelagatran plus acetylsalicylic acid (ASA) and clopidogrel plus ASA in a human ex vivo arterial thrombosis model

It was the objective of this study to compare the antithrombotic effects and bleeding profiles of the oral direct thrombin inhibitor ximelagatran, an anticoagulant, and the antiplatelet agent clopidogrel on top of steady-state acetylsalicylic acid (ASA) in a human arterial thrombosis model. Healthy male volunteers (n=62) received ASA (160 mg once daily), plus either clopidogrel for 6 days (loading dose 300 mg, then 75 mg once daily), or a single dose of ximelagatran (36 or 72 mg) on Day 6. Changes in total thrombus area (TTA) under low shear rate (LSR; 212 s(-1)) and high shear rate (HSR; 1690 s(-1)) conditions were measured, using the ex vivo Badimon perfusion chamber model pre-dose and 2 and 5 hours after dosing on Day 6, and capillary bleeding times (CBT) were determined. Ximelagatran plus ASA significantly reduced TTA under LSR and HSR, compared with ASA alone. Ximelagatran plus ASA reduced TTA more than clopidogrel plus ASA under LSR after 2 hours (36 mg, P=0.0011; 72 mg, P<0.0001) and 5 hours (72 mg, P=0.0057), and under HSR after 2 and 5 hours (72 mg, P<0.05). Compared with ASA alone, CBT was markedly prolonged by clopidogrel plus ASA (ratio 6.4; P<0.0001) but only slightly by ximelagatran plus ASA (72 mg ximelagatran, ratio 1.4; P=0.0010). Both drug combinations were well tolerated. Oral ximelagatran plus ASA has a greater antithrombotic effect in this human ex vivo thrombosis model and a less pronounced prolongation of bleeding time than clopidogrel plus ASA.

Mechanism of Action of the Oral Direct Thrombin Inhibitor Ximelagatran

Thrombin plays a central role in thrombus formation through its conversion of fibrinogen to fibrin and activation of platelets as well as amplifying its own generation by feedback activation via factors V, VIII, and XI. Consequently, thrombin represents a logical and promising target for therapeutic interventions against arterial and venous thromboembolic disorders. Ximelagatran is the first oral agent in the new class of direct thrombin inhibitors and is rapidly absorbed and bioconverted to the active moiety, melagatran, which inhibits fluid-phase and clot-bound thrombin with similar high potency. Binding to the active site of thrombin is direct and competitive and does not require the presence of co-factors. Inhibition of thrombin generation and platelet activation has been demonstrated in vitro with melagatran as well as ex vivo after oral administration of ximelagatran to healthy human volunteers. Oral ximelagatran dose dependently reduced the total thrombus area in an ex vivo flow chamber model of arterial thrombosis, reflecting the cumulative effect of inhibition of thrombin activity, thrombin generation, and platelet activation. Melagatran has also been shown to reduce thrombin-mediated inflammation in vitro. The combination of antithrombotic and anti-inflammatory activity with the practicality of oral dosing provided by ximelagatran represents an important new option for the treatment of arterial and venous thromboembolic disorders.

Acute antithrombotic effects of ximelagatran, an oral direct thrombin inhibitor, and r-hirudin in a human ex vivo model of arterial thrombosis

Thrombin plays a major role in thrombus formation through activation of platelets and conversion of fibrinogen to fibrin. To investigate the antithrombotic effects of the oral direct thrombin inhibitor (DTI) ximelagatran and the parenteral DTI r-hirudin in humans. Healthy male volunteers randomized into four parallel groups each with 15 subjects received either ximelagatran (20, 40 or 80 mg orally) or r-hirudin (0.4 mg kg-1 intravenous bolus + infusion of 0.15 mg kg-1 h-1 for 2 h and 0.075 mg kg-1 h-1 for 3 h). Antithrombotic effects were assessed as changes in total thrombus area (TTA) and total fibrin area (TFA) from baseline, using the Badimon perfusion chamber model at baseline and 2 h and 5 h after drug administration. Two hours postdosing, ximelagatran showed antithrombotic effects at both high and low shear rates (TTA% of mean baseline value +/- SEM was 76 +/- 13% and 71 +/- 17% [both P < 0.05] for the 20-mg dose, 85 +/- 11% [P > 0.05] and 62 +/- 15% [P < 0.05] for the 40-mg dose and 60 +/- 11% and 26 +/- 7% [both P < 0.05] for the 80-mg dose, respectively). r-Hirudin also showed a significant antithrombotic effect at high and low shear rates (76 +/- 11% [P = 0.05] and 57 +/- 17% [P < 0.05] of baseline values, 2 h postdosing, respectively). The inhibitory effects on TFA were similar to those on TTA. The oral DTI ximelagatran shows antithrombotic effects under both high and low shear conditions. The antithrombotic effect of 40-80 mg ximelagatran appeared comparable to that of parenterally administered r-hirudin, which has been previously demonstrated to be clinically effective in acute coronary syndromes.

Antithrombotic Effect of Tissue Factor Inhibition by Inactivated Factor VIIa

FFR-rFVIIa is an inactivated recombinant factor VIIa (rFVIIa) that inhibits the binding of factor VIIa to tissue factor (TF). It has been shown to prevent TF-induced thrombosis in animals. The present study is a substudy of the Active Site Inhibited Seven (ASIS) trial and examines the antithrombotic effect of 3 doses of FFR-rFVIIa in 24 patients undergoing percutaneous coronary intervention (PCI). Group 1 (n=9) received 400 microg/kg FFR-rFVIIa and 40 to 50 U/kg heparin, group 2 (n=7) received 200 microg/kg FFR-rFVIIa and 100 U/kg heparin, and group 3 (n=8) received 50 microg/kg FFR-rFVIIa and 100 U/kg heparin. Blood thrombogenicity was assessed as total thrombus area and fibrin deposition on the perfusion chamber at shear rate conditions typical of mild-moderate coronary stenosis. Baseline blood thrombogenicity was evaluated a day before PCI, after heparin administration. A second perfusion chamber study was performed just before PCI, 15 minutes after the administration of heparin and FFR-rFVIIa. Thrombus formation at a high shear rate was markedly reduced in groups 1 and 2 after drug administration, by 79% to 84% and 76% to 87%, respectively (P<0.004 [group 1], P<0.04 [group 2]). In group 3, moderate thrombus reduction of 46% to 48% was achieved (P<0.04). Fibrin deposition in all 3 groups was nearly eliminated after drug administration. Our data demonstrate that FFR-rFVIIa has a potent antithrombotic effect at different shear rates and severe arterial injury conditions.

Neovascularization during venous thrombosis organization: A preliminary study

Purpose: Thrombus organization after venous thromboembolism leading to recanalization occurs at a variable rate. The angiogenic chemokine interleukin-8 (IL-8) has been found in thrombus months after thrombus initiation. We hypothesize that thrombus organization involves neovascularization and leukocyte influx and that IL-8 administered at thrombus induction will promote thrombus organization. Methods: A group of rats underwent inferior vena caval occlusive thrombosis. At thrombus induction and every 24 hours, the rats were administered IL-8 (1 μg) or serum albumin. The rats were killed at either day 4, day 8, or day 12, and, at death, colloidal carbon was perfused via the heart. The inferior vena cava was isolated, measured, weighed, and formalin fixed. The sections were stained with anti-polymorphonuclear leukocyte antibody, the endothelial marker factor VIII–related antigen, and with hematoxylin and eosin. Thrombus neovascularization (colloidal carbon) with morphometric analysis was normalized to the total thrombus area. In addition, the rats underwent perfusion with fluorescein isothiocyanate dextran (molecular weight, 150,000) at death to correlate with colloidal carbon perfusion, and thrombus fluorescence was determined. Results: Thrombus cellularity initially involved neutrophils, followed by monocytes. Significantly more neutrophils, monocytes, and cells that were defined as spindle shaped (fibroblasts and endothelial cells) were noted in the animals treated with IL-8. Neovascularization was significantly increased at day 4 in the animals treated with IL-8 versus the animals treated with serum albumin and was corroborated with a significant increase in thrombus fluorescein isothiocyanate dextran fluorescence at day 4 in the rats treated with IL-8. Colloidal carbon perfusion was noted within vascular channels without extravasation and colocalized with factor VIII–related antigen. Conclusion: This study shows that thrombus organization involves neovascularization and that IL-8 augments thrombus organization. (J Vasc Surg 1999;30:885-93.)

Evaluation of the thrombogenecity of microvascular prosthesis by in vivo microscopy.

Expanded polytetrafluoroethylene(ePTFE) grafts 4mm long and 1mm in diameter were implanted into the iliac artery of 100-150g male rats using standard microvascular technique. Prior to clamp removal, the cremaster muscle was isolated as an island flap based on the iliac artery and observed using intravital fluorescence microscopy. Fields which contained a bifurcation of a first order arteriole(80-100 microns diameter) into second order arteriole(50-80 microns) were chosen for observation. Platelets were labeled in vivo with acridine red to visualize and quantify the aggregates. Images of microemboli were counted manually and the area was measured by computerized planimetry. Six control grafts were implanted with no further processing, six were irrigated with heparin, and six were coated with tridodecylmethylammonium chloride(TDMAC) and heparin. Most thrombi appeared within the first five minutes after implantation in all groups. The total number of emboli observed in the control group was 91 pr animal, in the heparin irrigation group it was 84, and in the TDMAC-heparin group it was 22. The total thrombus area observed per animal was 137,660 +/- 29,467 microns 2 in the control group, 79,040 +/- 10,893 microns 2 in the heparin irrigation group, and 17,498 +/- 6,059 microns 2 in the TDMAC-heparin group (p < .01 vs control or heparin irrigation group). With this results we could find that heparin irrigation and TDMAC-heparin coating appear to reduce the number, size, and total amount of microemboli generated by ePTFE graft implantation and apparent thromboresistant property of TDMAC-heparin coating may have widespread application in many clinical and research areas and this experimental model can be used for evaluation of other graft matrials.