Key Points• Thrombus formation and contraction alters local molecular transport, which regulates agonist distribution and platelet activation.• Semaphorin 4D contactdependent signaling increases platelet activation, but does not affect platelet packing or agonist transport.Hemostatic thrombi develop a characteristic architecture in which a core of highly activated platelets is covered by a shell of less-activated platelets. Here we have used a systems biology approach to examine the interrelationship of this architecture with transport rates and agonist distribution in the gaps between platelets. Studies were performed in mice using probes for platelet accumulation, packing density, and activation plus recently developed transport and thrombin activity probes. The results show that intrathrombus transport within the core is much slower than within the shell. The region of slowest transport coincides with the region of greatest packing density and thrombin activity, and appears prior to full platelet activation. Deleting the contact-dependent signaling molecule, Sema4D, delays platelet activation, but not the emergence of the low transport region. Collectively, these results suggest a timeline in which initial platelet accumulation and the narrowing gaps between platelets create a region of reduced transport that facilitates local thrombin accumulation and greater platelet activation, whereas faster transport rates within the shell help to limit thrombin accumulation and growth of the core. Thus, from a systems perspective, platelet accumulation produces an altered microenvironment that shapes thrombus architecture, which in turn affects agonist distribution and subsequent thrombus growth. (Blood. 2014;124(11):1808-1815 IntroductionThe hemostatic response balances the need to halt bleeding with the need to avoid inappropriate vascular occlusion. Recent reports of hemostatic thrombi formed in vivo have demonstrated that the extent of platelet activation within a growing thrombus is heterogeneous [1][2][3][4][5] and can result in a characteristic core-and-shell architecture. We have shown that the core region develops adjacent to the injury site and consists of fully activated, closely packed platelets that have undergone a-granule exocytosis, which allows them to be recognized by the appearance of the a-granule membrane protein, P-selectin, on their surface. 3 The shell is a less stable region that coats the core and consists of loosely packed, less activated platelets. 3Regional differences in the extent of platelet activation can potentially be driven by multiple factors. Here we have adopted a systems biology perspective, looking beyond the events in any one platelet to test the idea that the emerging architecture of the hemostatic response serves as both a driver and a reflection of differences in intrathrombus molecular transport rates and consequent differences in agonist distribution. Numerous platelet agonists are present during vascular injury, including collagen, thrombin, adenosine 59-diphosphate (ADP)...

Hemostatic thrombi formed after a penetrating injury have a distinctive structure in which a core of highly activated, closely packed platelets is covered by a shell of less-activated, loosely packed platelets. We have shown that differences in intrathrombus molecular transport emerge in parallel with regional differences in platelet packing density and predicted that these differences affect thrombus growth and stability. Here we test that prediction in a mouse vascular injury model. The studies use a novel method for measuring thrombus contraction in vivo and a previously characterized mouse line with a defect in integrin αIIbβ3 outside-in signaling that affects clot retraction ex vivo. The results show that the mutant mice have a defect in thrombus consolidation following vascular injury, resulting in an increase in intrathrombus transport rates and, as predicted by computational modeling, a decrease in thrombin activity and platelet activation in the thrombus core. Collectively, these data (1) demonstrate that in addition to the activation state of individual platelets, the physical properties of the accumulated mass of adherent platelets is critical in determining intrathrombus agonist distribution and platelet activation and (2) define a novel role for integrin signaling in the regulation of intrathrombus transport rates and localization of thrombin activity.

• Hindered diffusion becomes the dominant force of molecular movement in a thrombus.• The thrombus core acts as a selective molecular prison.Hemostatic thrombi formed after a penetrating injury have a heterogeneous architecture in which a core of highly activated, densely packed platelets is covered by a shell of lessactivated, loosely packed platelets. In the first manuscript in this series, we show that regional differences in intrathrombus protein transport rates emerge early in the hemostatic response and are preserved as the thrombus develops. Here, we use a theoretical approach to investigate this process and its impact on agonist distribution. The results suggest that hindered diffusion, rather than convection, is the dominant mechanism responsible for molecular movement within the thrombus. The analysis also suggests that the thrombus core, as compared with the shell, provides an environment for retaining soluble agonists such as thrombin, affecting the extent of platelet activation by establishing agonist-specific concentration gradients radiating from the site of injury. This analysis accounts for the observed weaker activation and relative instability of platelets in the shell and predicts that a failure to form a tightly packed thrombus core will limit thrombin accumulation, a prediction tested by analysis of data from mice with a defect in clot retraction. (Blood.

The electrical activity pattern of endocrine pituitary cells regulates their basal secretion level. Rat somatotrophs and lactotrophs exhibit spontaneous bursting and have high basal levels of hormone secretion, while gonadotrophs exhibit spontaneous spiking and have low basal hormone secretion. It has been proposed that the difference in electrical activity between bursting somatotrophs and spiking gonadotrophs is due to the presence of large conductance potassium (BK) channels on somatotrophs but not on gonadotrophs. This is one example where the role of an ion channel type may be clearly established. We demonstrate here that BK channels indeed promote bursting activity in pituitary cells. Blocking BK channels in bursting lacto-somatotroph GH4C1 cells changes their firing activity to spiking, while further adding an artificial BK conductance via dynamic clamp restores bursting. Importantly, this burst-promoting effect requires a relatively fast BK activation/deactivation, as predicted by computational models. We also show that adding a fast activating BK conductance to spiking gonadotrophs converts the activity of these cells to bursting. Together, our results suggest that differences in BK channel expression may underlie the differences in electrical activity and basal hormone secretion levels among pituitary cell types and that the rapid rate of BK channel activation is key to its role in burst promotion.

Hemostasis requires the tightly regulated interaction of the coagulation system, platelets, other blood cells and components of the vessel wall at a site of vascular injury. The dysregulation of this response may result in excessive bleeding if the response is impaired, and pathologic thrombosis with vessel occlusion and tissue ischemia if the response is overly robust. Extensive studies over several decades have elucidated the major molecular signaling pathways responsible for platelet activation and aggregation, and anti-thrombotic agents targeting several of these pathways are in widespread clinical use. This review will summarize more recent research examining mechanisms by which these multiple platelet signaling pathways are integrated in time and space at a site of vascular injury in vivo to produce an optimal hemostatic response.

Background Hemorrhagic shock is a leading cause of death following severe trauma and platelet transfusions are frequently necessary to achieve hemostasis. Platelets, however, require special storage conditions and storage time has been associated with loss of platelet quality. We hypothesized that standard storage conditions have a deleterious effect on platelet mitochondrial function and platelet activation. Materials and methods Platelet donations were collected from healthy donors (n=5), and stored in gas permeable collection bags according to American Association of Blood Bank recommendations. Platelet units were sampled from day of collection (day 0) until day 7. High resolution respirometry was used to assess baseline mitochondrial respiration, maximal oxygen utilization, and individual mitochondrial complex-dependent respiration. FACS was performed to analyze mitochondrial content, mitochondrial reactive oxygen species (ROS), the expression of P-selectin (both before and after challenge with thrombin receptor activating peptide (SFLLRN)), and apoptosis. Data was analyzed using ANOVA and Pearson’s correlation (p<0.05 significant). Results Mitochondrial respiration decreased significantly in platelets stored longer than 2 days (p<0.05). Platelets also demonstrated a persistent decrease in response to stimulation with SFLLRN by the 3rd day of storage (p<0.05) as well as an increase in mitochondrial ROS and apoptosis (p<0.05). Mitochondrial respiration significantly correlated with platelet capacity to activate (r=0.8, p<0.05). Conclusion Platelet mitochondrial respiratory function and activation response decrease significantly in platelets stored for 3 or more days. Because platelet transfusions almost universally occur between the 3rd and 5th day of storage, our findings may have significant clinical importance and warrant further in vivo analysis.

All-electronic DNA biosensors based on graphene field-effect transistors (GFETs) offer the prospect of simple and cost-effective diagnostics. For GFET sensors based on complementary probe DNA, the sensitivity is limited by the binding affinity of the target oligonucleotide, in the nM range for 20 mer targets. We report a ∼20 000× improvement in sensitivity through the use of engineered hairpin probe DNA that allows for target recycling and hybridization chain reaction. This enables detection of 21 mer target DNA at sub-fM concentration and provides superior specificity against single-base mismatched oligomers. The work is based on a scalable fabrication process for biosensor arrays that is suitable for multiplexed detection. This approach overcomes the binding-affinity-dependent sensitivity of nucleic acid biosensors and offers a pathway toward multiplexed and label-free nucleic acid testing with high accuracy and selectivity.

Extensive studies have detailed the molecular regulation of individual components of the hemostatic system, including platelets, coagulation factors, and regulatory proteins. Questions remain, however, about how these elements are integrated at the systems level within a rapidly changing physical environment. To answer some of these questions, we developed a puncture injury model in mouse jugular veins that combines high-resolution, multimodal imaging with functional readouts in vivo. The results reveal striking spatial regulation of platelet activation and fibrin formation that could not be inferred from studies performed ex vivo. As in the microcirculation, where previous studies have been performed, gradients of platelet activation are readily apparent, as is an asymmetrical distribution of fibrin deposition and thrombin activity. Both are oriented from the outer to the inner surface of the damaged vessel wall, with a greater extent of platelet activation and fibrin accumulation on the outside than the inside. Further, we show that the importance of P2Y12signaling in establishing a competent hemostatic plug is related to the size of the injury, thus limiting its contribution to hemostasis to specific physiologic contexts. Taken together, these studies offer insights into the organization of hemostatic plugs, provide a detailed understanding of the adverse bleeding associated with a widely prescribed class of antiplatelet agents, and highlight differences between hemostasis and thrombosis that may suggest alternative therapeutic approaches.