Peter L. Gross

Accumulation of Tissue Factor into Developing Thrombi In Vivo Is Dependent upon Microparticle P-Selectin Glycoprotein Ligand 1 and Platelet P-Selectin

Using a laser-induced endothelial injury model, we examined thrombus formation in the microcirculation of wild-type and genetically altered mice by real-time in vivo microscopy to analyze this complex physiologic process in a system that includes the vessel wall, the presence of flowing blood, and the absence of anticoagulants. We observe P-selectin expression, tissue factor accumulation, and fibrin generation after platelet localization in the developing thrombus in arterioles of wild-type mice. However, mice lacking P-selectin glycoprotein ligand 1 (PSGL-1) or P-selectin, or wild-type mice infused with blocking P-selectin antibodies, developed platelet thrombi containing minimal tissue factor and fibrin. To explore the delivery of tissue factor into a developing thrombus, we identified monocyte-derived microparticles in human platelet–poor plasma that express tissue factor, PSGL-1, and CD14. Fluorescently labeled mouse microparticles infused into a recipient mouse localized within the developing thrombus, indicating that one pathway for the initiation of blood coagulation in vivo involves the accumulation of tissue factor– and PSGL-1–containing microparticles in the platelet thrombus expressing P-selectin. These monocyte-derived microparticles bind to activated platelets in an interaction mediated by platelet P-selectin and microparticle PSGL-1. We propose that PSGL-1 plays a role in blood coagulation in addition to its known role in leukocyte trafficking.

Real-time in vivo imaging of platelets, tissue factor and fibrin during arterial thrombus formation in the mouse.

We have used confocal and widefield microscopy to image thrombus formation in real time in the microcirculation of a living mouse. This system provides high-speed, near-simultaneous acquisition of images of multiple fluorescent probes and of a brightfield channel. Vascular injury is induced with a laser focused through the microscope optics. We observed platelet deposition, tissue factor accumulation and fibrin generation after laser-induced endothelial injury in a single developing thrombus. The initiation of blood coagulation in vivo entailed the initial accumulation of tissue factor on the upstream and thrombus–vessel wall interface of the developing thrombus. Subsequently tissue factor was associated with the interior of the thrombus. Tissue factor was biologically active, and was associated with fibrin generation within the thrombus.

Leukocyte-versus microparticle-mediated tissue factor transfer during arteriolar thrombus development

Circulating tissue factor accumulates in the developing thrombus and contributes to fibrin clot formation. To determine whether tissue factor derived from hematopoietic cells is delivered to the thrombus via tissue factor‐bearing microparticles or circulating leukocytes expressing tissue factor on the plasma membrane, we compared the kinetics of tissue factor accumulation in the developing arteriolar thrombus with the time course of leukocyte‐thrombus interaction and microparticle‐thrombus interaction in the microcirculation of a living mouse using intravital high‐speed widefield and confocal microscopy. Tissue factor rapidly accumulated in the developing thrombus, appearing immediately following vessel wall injury, reaching a first peak in ∼100 s. In contrast, leukocyte‐thrombus interaction was not observed until after 2–3 min following vessel wall injury. Maximal leukocyte rolling and firm leukocyte adherence on thrombi in wild‐type mice were observed after ∼8 min and were dependent on P‐selectin and P‐selectin glycoprotein ligand‐1. This delay in P‐selectin‐dependent leukocyte rolling is a result of time‐dependent platelet activation and P‐selectin expression on the luminal surface of the thrombus. In contrast, microparticle accumulation in the developing arteriolar thrombus was rapid, and peak accumulation was within 60 s. The accumulation of hematopoietic cell‐derived tissue factor in the developing thrombus correlates to the kinetics of microparticle accumulation and does not correlate temporally with leukocyte‐thrombus interaction. These results indicate that tissue factor derived from hematopoietic cells is delivered by microparticles during the initial phase of thrombus development in vivo.

Thrombus formation: direct real-time observation and digital analysis of thrombus assembly in a living mouse by confocal and widefield intravital microscopy.

Summary.  We have developed novel instrumentation using confocal and widefield microscopy to image and analyze thrombus formation in real time in the microcirculation of a living mouse. This system provides high‐speed, near‐simultaneous acquisition of images of multiple fluorescent probes and a brightfield channel, and supports laser‐induced injury through the microscope optics. Although this imaging facility requires interface of multiple hardware components, the primary challenge in vascular imaging is careful experimental design and interpretation. This system has been used to localize tissue factor during thrombus formation, to observe defects in thrombus assembly in genetically altered mice, to study the kinetics of platelet activation and P‐selectin expression following vascular injury, to analyze leukocyte rolling on arterial thrombi, to generate three‐dimensional models of thrombi, and to analyze the effect of antithrombotic agents in vivo.

Vitronectin stabilizes thrombi and vessel occlusion but plays a dual role in platelet aggregation

Summary.  The role of vitronectin (Vn) in thrombosis is currently controversial; both inhibitory and supportive roles have been reported. To monitor directly the function of Vn in thrombotic events at the site of vascular injury, we studied Vn‐deficient (Vn–/–) and wild‐type (WT) control mice with two real‐time intravital microscopy thrombosis models. In the mesenteric arteriole model, vessel injury was induced by ferric chloride. We observed unstable thrombi and a significantly greater number of emboli in Vn–/– mice. Vessel occlusion was also delayed and frequent vessel re‐opening occurred. In the cremaster muscle arteriole model, vessel injury was induced by a nitrogen dye laser. We observed significantly fewer platelets, lower fibrin content, and unstable fibrin within the thrombi of Vn–/– mice. To define further the role of Vn in thrombus growth, we studied platelet aggregation in vitro. Consistent with our in vivo data, the second wave of thrombin‐induced aggregation of gel‐filtered platelets was abolished at a low concentration of thrombin in Vn–/– platelets. Interestingly, adenosine diphosphate (ADP)‐induced platelet aggregation was significantly increased in Vn–/– platelet‐rich plasma (PRP) and this effect was attenuated by adding purified plasma Vn. We also observed increased platelet aggregation induced by shear stress in Vn–/– whole blood. These data demonstrate that Vn is a thrombus stabilizer. However, in contrast to released platelet granule Vn which enhances platelet aggregation, plasma Vn inhibits platelet aggregation.

Reduced Agonist-Induced Endothelium-Dependent Vasodilation in Uremia Is Attributable to an Impairment of Vascular Nitric Oxide

Current concepts for the explanation of endothelial dysfunction and accelerated atherosclerosis in uremia propose a reduced vascular bioavailability of nitric oxide (NO). The aim of the present study was to test the contributions of NO and NO/prostacyclin (PGI(2))-independent mechanisms to both baseline vascular tone and agonist-induced endothelium-dependent vasodilation in patients on hemodialysis (HD). In 10 HD patients and eight matched healthy control subjects, forearm blood flow (FBF) was measured at rest and during intrabrachial infusions of norepinephrine (NE; endothelium-independent vasoconstrictor, 60, 120, and 240 pmol/min) and N-monomethyl-L-arginine (blocker of NO synthases, 16 micromol/min). After inhibition of cyclo-oxygenase by ibuprofen (1200 mg orally), endothelium-dependent and -independent vasodilation was assessed by infusion of acetylcholine (ACh; 1, 5, 10, 50, 100, and 300 nmol/min) and sodium-nitroprusside (2.5, 5, and 10 microg/min). NO/PGI(2)-independent vasodilation was tested by equal infusions of ACh during NO clamp. N-monomethyl-L-arginine reduced resting FBF to a comparable degree in both groups. Vascular responses to ACh were reduced in HD (P = 0.003 versus control by ANOVA), whereas those to sodium nitroprusside were mainly at control level. Infusion of ACh during NO clamp caused a similar increment of FBF in both groups. NO-mediated vasodilation as calculated by the difference between ACh-induced responses without and with NO clamp was substantially impaired in HD (P < 0.001) compared with control. In HD patients, baseline NO-mediated arteriolar tone is at control level. This study provides first evidence that endothelial dysfunction of uremic patients as shown by reduced agonist-induced endothelium-dependent vasodilation is attributable to reduced stimulation of NO, whereas the NO/PGI(2)-resistant portion of ACh-mediated vasodilation is unaffected.

Aspirin versus low-molecular-weight heparin for extended venous thromboembolism prophylaxis after total hip arthroplasty: a randomized trial.

BACKGROUND The role of aspirin in thromboprophylaxis after total hip arthroplasty (THA) is controversial. OBJECTIVE To compare extended prophylaxis with aspirin and dalteparin for prevention of symptomatic venous thromboembolism (VTE) after THA. DESIGN Multicenter randomized, controlled trial with a noninferiority design based on a minimal clinically important difference of 2.0%. Randomization was electronically generated; patients were assigned to a treatment group through a Web-based program. Patients, physicians, study coordinators, health care team members, outcome adjudicators, and data analysts were blinded to interventions. (Current Controlled Trials: ISRCTN11902170). SETTING 12 tertiary care orthopedic referral centers in Canada. PATIENTS 778 patients who had elective unilateral THA between 2007 and 2010. INTERVENTION After an initial 10 days of dalteparin prophylaxis after elective THA, patients were randomly assigned to 28 days of dalteparin (n = 400) or aspirin (n = 386). MEASUREMENTS Symptomatic VTE confirmed by objective testing (primary efficacy outcome) and bleeding. RESULTS Five of 398 patients (1.3%) randomly assigned to dalteparin and 1 of 380 (0.3%) randomly assigned to aspirin had VTE (absolute difference, 1.0 percentage point [95% CI, -0.5 to 2.5 percentage points]). Aspirin was noninferior (P < 0.001) but not superior (P = 0.22) to dalteparin. Clinically significant bleeding occurred in 5 patients (1.3%) receiving dalteparin and 2 (0.5%) receiving aspirin. The absolute between-group difference in a composite of all VTE and clinically significant bleeding events was 1.7 percentage points (CI, -0.3 to 3.8 percentage points; P = 0.091) in favor of aspirin. LIMITATION The study was halted prematurely because of difficulty with patient recruitment. CONCLUSION Extended prophylaxis for 28 days with aspirin was noninferior to and as safe as dalteparin for the prevention of VTE after THA in patients who initially received dalteparin for 10 days. Given its low cost and greater convenience, aspirin may be considered a reasonable alternative for extended thromboprophylaxis after THA. PRIMARY FUNDING SOURCE Canadian Institutes of Health Research.

Evolving use of new oral anticoagulants for treatment of venous thromboembolism

The new oral anticoagulants (NOACs), which include dabigatran, rivaroxaban, apixaban, and edoxaban, are poised to replace warfarin for treatment of the majority of patients with venous thromboembolism (VTE). With a rapid onset of action and the capacity to be administered in fixed doses without routine coagulation monitoring, NOACs streamline VTE treatment. In phase 3 trials in patients with acute symptomatic VTE, NOACs have been shown to be noninferior to conventional anticoagulant therapy for prevention of recurrence and are associated with less bleeding. Rivaroxaban and dabigatran are already licensed for VTE treatment in the United States, and apixaban and edoxaban are under regulatory consideration for this indication. As the number of approved drugs increases, clinicians will need to choose the right anticoagulant for the right VTE patient. To help with this decision, this review (1) compares the pharmacologic profiles of the NOACs, (2) outlines the unique design features of the phase 3 trials that evaluated the NOACs for VTE treatment, (3) reviews the results of these trials highlighting similarities and differences in the findings, (4) provides perspective about which VTE patients should receive conventional treatment or are candidates for NOACs, and (5) offers suggestions about how to choose among the NOACs.

New oral anticoagulants: which one should my patient use?

The new oral anticoagulants are rapidly replacing warfarin for several indications. In contrast to warfarin, which lowers the functional levels of all of the vitamin K-dependent clotting factors, the new agents target either factor Xa or thrombin. With targeted inhibition of coagulation, the new oral anticoagulants have pharmacologic and clinical features that distinguish them from warfarin. Focusing on these features, this paper (a) compares the pharmacology of the new oral anticoagulants with that of warfarin (b) identifies the class effects of these drugs and their differentiating features, (c) reviews their current indications, and (d) uses this information to help clinicians make informed decisions regarding the choice of the right anticoagulant for the right patient.

Delayed but not Early Treatment with DNase Reduces Organ Damage and Improves Outcome in a Murine Model of Sepsis.

ABSTRACT Sepsis is characterized by systemic activation of coagulation and inflammation in response to microbial infection. Although cell-free DNA (cfDNA) released from activated neutrophils has antimicrobial properties, it may also exert harmful effects by activating coagulation and inflammation. The authors aimed to determine whether deoxyribonuclease (DNase) administration reduces cfDNA levels, attenuates coagulation and inflammation, suppresses organ damage, and improves outcome in a cecal ligation and puncture (CLP) model of polymicrobial sepsis. Healthy C57Bl/6 mice were subjected to CLP, a surgical procedure involving two punctures of the ligated cecum, or sham surgery (no ligation/puncture). Mice were given DNase or saline by intraperitoneal injection 2, 4, or 6 h after surgery. Two hours after treatment, organs were harvested and plasma levels of cfDNA, interleukin-6 (IL-6), IL-10, thrombin-antithrombin complexes, lung myeloperoxidase, creatinine, alanine transaminase, and bacterial load were quantified. Survival studies were also performed. The CLP-operated mice had rapid time-dependent elevations in cfDNA that correlated with elevations in IL-6, IL-10, and thrombin-antithrombin complexes and had organ damage in the lungs and kidneys. Administration of DNase at 2 h after CLP resulted in increased IL-6 and IL-10 levels and organ damage in the lungs and kidneys. In contrast, DNase administration at 4 or 6 h after CLP resulted in reduced cfDNA and IL-6 levels, increased IL-10, and suppressed organ damage and bacterial dissemination. Deoxyribonuclease administration every 6 h after CLP also rescued mice from death. Our studies are the first to demonstrate that delayed but not early administration of DNase may be protective in experimental sepsis.