Development of a method to quantify platelet adhesion and aggregation under static conditions.

Platelets are important players in hemostasis and thrombosis. Thus, accurate assessment of platelet function is crucial for identifying platelet function disorders and measuring the efficacy of antiplatelet therapies. We have developed a novel platelet aggregation technique that utilizes the physical parameter of platelet concentration in conjunction with volume and mass measurements to evaluate platelet adhesion and aggregation. Platelet aggregates were formed by incubating purified platelets on fibrinogen- or fibrillar collagen-coated surfaces at platelet concentrations ranging from 20,000 to 500,000 platelets/ L. Platelets formed aggregates under static conditions in a platelet concentration-dependent manner, with significantly greater mean volume and mass at higher platelet concentrations ( 400,000 platelets/ L). We show that a platelet glycoprotein IIb/IIIa inhibitor abrogated platelet-platelet aggregation, which significantly reduced the volume and mass of the platelets on the collagen surface. This static platelet aggregation technique is amenable to standardization and represents a useful tool to investigate the mechanism of platelet activation and aggregation under static conditions.

Measurement science in the circulatory system.

The dynamics of the cellular and molecular constituents of the circulatory system are regulated by the biophysical properties of the heart, vasculature and blood cells and proteins. In this review, we discuss measurement techniques that have been developed to characterize the physical and mechanical parameters of the circulatory system across length scales ranging from the tissue scale (centimeter) to the molecular scale (nanometer) and time scales of years to milliseconds. We compare the utility of measurement techniques as a function of spatial resolution and penetration depth from both a diagnostic and research perspective. Together, this review provides an overview of the utility of measurement science techniques to study the spatial systems of the circulatory system in health and disease.

Stimulus-evoked calcium transients in somatosensory cortex are temporarily inhibited by a nearby microhemorrhage.

Although microhemorrhages are common in the brain of the elderly, the direct impact of these lesions on neural function remains unclear. In this work, we used femtosecond laser irradiation to rupture the wall of single arterioles in the brain of anesthetized rodents, producing a hematoma of ∼100-µm diameter. Our objective was to study the impact of these microhemorrhages on cortical activity using cell-resolved two-photon imaging of bulk-loaded calcium-sensitive dye. We monitored peripheral sensory stimulus-induced calcium transients from individual neuronal cell bodies, regions of neuropil, and astrocytes at different distances from the microhemorrhage before and 0.5, 2, and 4 hours after the creation of the lesion. We found that immediately after the hemorrhage the average amplitude of the stimulus-induced calcium response was reduced to about half within 150 µm from the hematoma. Beyond 300 µm, there was little effect on cell response, with a smooth increase in response amplitude from 150 µm to 300 µm from the lesion. Cortical function gradually improved with time and by four hours after the lesion the response from neurons and astrocytes had recovered to baseline everywhere but within 150 µm from the hematoma. To assess whether the cells closest to the microhemorrhage recovered over a longer timeframe, we developed a re-openable chronic cranial window preparation that allowed reinjection of calcium-sensitive fluorescent dye. We found that the response largely recovered by one day after the microhemorrhage even within 150 µm from the hematoma. This work suggests that neuronal and astrocyte function is transiently lost near a microhemorrhage, but recovers within one day after the lesion.

Intravenous tPA therapy does not worsen acute intracerebral hemorrhage in mice.

Tissue plasminogen activator (tPA) is the only FDA-approved treatment for reperfusing ischemic strokes. But widespread use of tPA is still limited by fears of inadvertently administering tPA in patients with intracerebral hemorrhage (ICH). Surprisingly, however, the assumption that tPA will worsen ICH has never been biologically tested. Here, we assessed the effects of tPA in two models of ICH. In a mouse model of collagenase-induced ICH, hemorrhage volumes and neurological deficits after 24 hrs were similar in saline controls and tPA-treated mice, whereas heparin-treated mice had 3-fold larger hematomas. In a model of laser-induced vessel rupture, tPA also did not worsen hemorrhage volumes, while heparin did. tPA is known to worsen neurovascular injury by amplifying matrix metalloproteinases during cerebral ischemia. In contrast, tPA did not upregulate matrix metalloproteinases in our mouse ICH models. In summary, our experimental data do not support the assumption that intravenous tPA has a deleterious effect in acute ICH. However, due to potential species differences and the inability of models to fully capture the dynamics of human ICH, caution is warranted when considering the implications of these findings for human therapy.

Development of coagulation factor probes for the identification of procoagulant circulating tumor cells.

Metastatic cancer is associated with a hypercoagulable state, and pathological venous thromboembolic disease is a significant source of morbidity and the second leading cause of death in patients with cancer. Here we aimed to develop a novel labeling strategy to detect and quantify procoagulant circulating tumor cells (CTCs) from patients with metastatic cancer. We hypothesize that the enumeration of procoagulant CTCs may be prognostic for the development of venous thrombosis in patients with cancer. Our approach is based on the observation that cancer cells are capable of initiating and facilitating cell-mediated coagulation in vitro, whereby activated coagulation factor complexes assemble upon cancer cell membrane surfaces. Binding of fluorescently labeled, active site-inhibited coagulation factors VIIa, Xa, and IIa to the metastatic breast cancer cell line, MDA-MB-231, non-metastatic colorectal cell line, SW480, or metastatic colorectal cell line, SW620, was characterized in a purified system, in anticoagulated blood and plasma, and in plasma under conditions of coagulation. We conclude that a CTC labeling strategy that utilizes coagulation factor-based fluorescent probes may provide a functional assessment of the procoagulant potential of CTCs, and that this strategy is amenable to current CTC detection platforms.

Anticoagulation with the oral direct thrombin inhibitor dabigatran does not enlarge hematoma volume in experimental intracerebral hemorrhage.

BACKGROUND: The direct thrombin inhibitor dabigatran etexilate (DE) may constitute a future replacement of vitamin K antagonists for long-term anticoagulation. Whereas warfarin pretreatment is associated with greater hematoma expansion after intracerebral hemorrhage (ICH), it remains unclear what effect direct thrombin inhibitors would have. Using different experimental models of ICH, this study compared hematoma volume among DE-treated mice, warfarin-treated mice, and controls. METHODS AND RESULTS: CD-1 mice were fed with DE or warfarin. Sham-treated mice served as controls. At the time point of ICH induction, DE mice revealed an increased activated partial thromboplastin time compared with controls (mean±SD 46.1 ± 5.0 versus 18.0 ± 1.5 seconds; P=0.022), whereas warfarin pretreatment resulted in a prothrombin time prolongation (51.4 ± 17.9 versus 10.4 ± 0.3 seconds; P<0.001). Twenty-four hours after collagenase-induced ICH formation, hematoma volume was 3.8 ± 2.9 µL in controls, 4.8 ± 2.7 µL in DE mice, and 14.5 ± 11.8 µL in warfarin mice (n=16; Welch ANOVA between-group differences P=0.007; posthoc analysis with the Dunnett method: DE versus controls, P=0.899; warfarin versus controls, P<0.001; DE versus warfarin, P=0.001). In addition, a model of laser-induced cerebral microhemorrhage was applied, and the distances that red blood cells and blood plasma were pushed into the brain were quantified. Warfarin mice showed enlarged red blood cell and blood plasma diameters compared to controls, but no difference was found between DE mice and controls. CONCLUSIONS: In contrast with warfarin, pretreatment with DE did not increase hematoma volume in 2 different experimental models of ICH. In terms of safety, this observation may represent a potential advantage of anticoagulation with DE over warfarin.

Anticipatory control of motion-to-force transitions with the fingertips adapts optimally to task difficulty.

Moving our fingertips toward objects to produce well-directed forces immediately upon contact is fundamental to dexterous manipulation. This apparently simple motion-to-force transition in fact involves a time-critical, predictive switch in control strategy. Given that dexterous manipulation must accommodate multiple mechanical conditions, we investigated whether and how this transition adapts to task difficulty. Eight adults (19-39 yr) produced ramps of isometric vertical fingertip force against a rigid surface immediately following a tapping motion. By changing target surface friction and size, we defined an easier (sandpaper, 11 mm diam) versus a more difficult (polished steel, 5 mm diam) task. As in prior work, we assembled fine-wire electromyograms from all seven muscles of the index finger into a seven-dimensional vector defining the full muscle coordination pattern-and quantified its temporal evolution as its alignment with a reference coordination pattern vector for steady-state force production. As predicted by numerical optimizations to neuromuscular delays, our empirical and sigmoidal nonlinear regression analyses show that the coordination pattern transitions begin sooner for the more difficult tasks than for the easier tasks ( approximately 120 ms, P < 0.02, and approximately 115 ms, P < 0.015, respectively) and that the coordination pattern transition in alignment is well represented by a sigmoidal trend (R;2 > 0.7 in most cases). Importantly, the force vector following contact had smaller directional error (P < 0.02) for the more difficult task even though the transition in coordination pattern was less stereotypical and uniform than for the easier task. These adaptations of transition strategy to task difficulty are compatible with an optimization to counteract neuromuscular delays and noise to enable this fundamental element of dexterous manipulation.