Platelet packing density is an independent regulator of the hemostatic response to injury.

Essentials Platelet packing density in a hemostatic plug limits molecular movement to diffusion. A diffusion-dependent steep thrombin gradient forms radiating outwards from the injury site. Clot retraction affects the steepness of the gradient by increasing platelet packing density. Together, these effects promote hemostatic plug core formation and inhibit unnecessary growth. SUMMARY: Background Hemostasis studies performed in vivo have shown that hemostatic plugs formed after penetrating injuries are characterized by a core of highly activated, densely packed platelets near the injury site, covered by a shell of less activated and loosely packed platelets. Thrombin production occurs near the injury site, further activating platelets and starting the process of platelet mass retraction. Tightening of interplatelet gaps may then prevent the escape and exchange of solutes. Objectives To reconstruct the hemostatic plug macro- and micro-architecture and examine how platelet mass contraction regulates solute transport and solute concentration in the gaps between platelets. Methods Our approach consisted of three parts. First, platelet aggregates formed in vitro under flow were analyzed using scanning electron microscopy to extract data on porosity and gap size distribution. Second, a three-dimensional (3-D) model was constructed with features matching the platelet aggregates formed in vitro. Finally, the 3-D model was integrated with volume and morphology measurements of hemostatic plugs formed in vivo to determine how solutes move within the platelet plug microenvironment. Results The results show that the hemostatic mass is characterized by extremely narrow gaps, porosity values even smaller than previously estimated and stagnant plasma velocity. Importantly, the concentration of a chemical species released within the platelet mass increases as the gaps between platelets shrink. Conclusions Platelet mass retraction provides a physical mechanism to establish steep chemical concentration gradients that determine the extent of platelet activation and account for the core-and-shell architecture observed in vivo.

The small-molecule MERTK inhibitor UNC2025 decreases platelet activation and prevents thrombosis.

Essentials Signaling by Gas6 through Tyro3/Axl/Mer receptors is essential for stable platelet aggregation. UNC2025 is a small molecule inhibitor of the Mer tyrosine kinase. UNC2025 decreases platelet activation in vitro and thrombus formation in vivo. UNC2025's anti-platelet effect is synergistic with inhibition of the ADP receptor, P2Y12 . SUMMARY: Background Growth arrest-specific protein 6 signals through the TAM (TYRO-3-AXL-MERTK) receptor family, mediating platelet activation and thrombus formation via activation of the aggregate-stabilizing αIIb ß3 integrin. Objective To describe the antithrombotic effects mediated by UNC2025, a small-molecule MERTK tyrosine kinase inhibitor. Methods MERTK phosphorylation and downstream signaling were assessed by immunoblotting. Light transmission aggregometry, flow cytometry and microfluidic analysis were used to evaluate the impact of MERTK inhibition on platelet activation and stability of aggregates in vitro. The effects of MERTK inhibition on arterial and venous thrombosis, platelet accumulation at microvascular injury sites and tail bleeding times were determined with murine models. The effects of combined treatment with ADP-P2Y1&12 pathway antagonists and UNC2025 were also evaluated. Results and Conclusions Treatment with UNC2025 inhibited MERTK phosphorylation and downstream activation of AKT and SRC, decreased platelet activation, and protected animals from pulmonary embolism and arterial thrombosis without increasing bleeding times. The antiplatelet effect of UNC2025 was enhanced in combination with ADP-P2Y1&12 pathway antagonists, and a greater than additive effect was observed when these two agents with different mechanisms of inhibition were coadministered. TAM kinase signaling represents a potential therapeutic target, as inhibition of this axis, especially in combination with ADP-P2Y pathway antagonism, mediates decreased platelet activation, aggregate stability, and thrombus formation, with less hemorrhagic potential than current treatment strategies. The data presented here also demonstrate antithrombotic activity mediated by UNC2025, a novel translational agent, and support the development of TAM kinase inhibitors for clinical applications.

Hierarchical organization of the hemostatic response to penetrating injuries in the mouse macrovasculature.

Essentials Methods were developed to image the hemostatic response in mouse femoral arteries in real time. Penetrating injuries produced thrombi consisting primarily of platelets. Similar to arterioles, a core-shell architecture of platelet activation occurs in the femoral artery. Differences from arterioles included slower platelet activation and reduced thrombin dependence. SUMMARY: Background Intravital studies performed in the mouse microcirculation show that hemostatic thrombi formed after penetrating injuries develop a characteristic architecture in which a core of fully activated, densely packed platelets is overlaid with a shell of less activated platelets. Objective Large differences in hemodynamics and vessel wall biology distinguish arteries from arterioles. Here we asked whether these differences affect the hemostatic response and alter the impact of anticoagulants and antiplatelet agents. Methods Approaches previously developed for intravital imaging in the mouse microcirculation were adapted to the femoral artery, enabling real-time fluorescence imaging despite the markedly thicker vessel wall. Results Arterial thrombi initiated by penetrating injuries developed the core-and-shell architecture previously observed in the microcirculation. However, although platelet accumulation was greater in arterial thrombi, the kinetics of platelet activation were slower. Inhibiting platelet ADP P2Y12 receptors destabilized the shell and reduced thrombus size without affecting the core. Inhibiting thrombin with hirudin suppressed fibrin accumulation, but had little impact on thrombus size. Removing the platelet collagen receptor, glycoprotein VI, had no effect. Conclusions These results (i) demonstrate the feasibility of performing high-speed fluorescence imaging in larger vessels and (ii) highlight differences as well as similarities in the hemostatic response in the macro- and microcirculation. Similarities include the overall core-and-shell architecture. Differences include the slower kinetics of platelet activation and a smaller contribution from thrombin, which may be due in part to the greater thickness of the arterial wall and the correspondingly greater separation of tissue factor from the vessel lumen.

Spatiotemporal regulation of coagulation and platelet activation during the hemostatic response in vivo.

The hemostatic response 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 the past decade have sought to unravel the regulatory mechanisms that coordinate the multiple biochemical and cellular responses in time and space to ensure that an optimal response to vascular damage is achieved. These studies have relied in part on advances in in vivo imaging techniques in animal models, allowing for the direct visualization of various molecular and cellular events in real time during the hemostatic response. This review summarizes knowledge gained with these in vivo imaging and other approaches that provides new insights into the spatiotemporal regulation of coagulation and platelet activation at a site of vascular injury.

Platelet-targeting sensor reveals thrombin gradients within blood clots forming in microfluidic assays and in mouse.

BACKGROUND: Thrombin undergoes convective and diffusive transport, making it difficult to visualize during thrombosis. We developed the first sensor capable of revealing inner clot thrombin dynamics. METHODS AND RESULTS: An N-terminal-azido thrombin-sensitive fluorescent peptide (ThS-P) with a thrombin-releasable quencher was linked to anti-CD41 using click chemistry to generate a thrombin-sensitive platelet binding sensor (ThS-Ab). Rapid thrombin cleavage of ThS-P (K(m) = 40.3 µm, k(cat) = 1.5 s(-1) ) allowed thrombin monitoring by ThS-P or ThS-Ab in blood treated with 2-25 pm tissue factor (TF). Individual platelets had > 20-fold more ThS-Ab fluorescence after clotting. In a microfluidic assay of whole blood perfusion over collagen ± linked TF (wall shear rate = 100 s(-1) ), ThS-Ab fluorescence increased between 90 and 450 s for 0.1-1 molecule-TF µm(-2) and co-localized with platelets near fibrin. Without TF, neither thrombin nor fibrin was detected on the platelet deposits by 450 s. Using a microfluidic device to control the pressure drop across a thrombus forming on a porous collagen/TF plug (521 s(-1) ), thrombin and fibrin were detected at the thrombus-collagen interface at a zero pressure drop, whereas 80% less thrombin was detected at 3200 Pa in concert with fibrin polymerizing within the collagen. With anti-mouse CD41 ThS-Ab deployed in a mouse laser injury model, the highest levels of thrombin arose between 40 and 160 s nearest the injury site where fibrin co-localized and where the thrombus was most mechanically stable. CONCLUSION: ThS-Ab reveals thrombin locality, which depends on surface TF, flow and intrathrombus pressure gradients.

Regulating thrombus growth and stability to achieve an optimal response to injury.

An optimal platelet response to injury can be defined as one in which blood loss is restrained and haemostasis is achieved without the penalty of further tissue damage caused by unwarranted vascular occlusion. This brief review considers some of the ways in which thrombus growth and stability can be regulated so that an optimal platelet response can be achieved in vivo. Three related topics are considered. The first focuses on intracellular mechanisms that regulate the early events of platelet activation downstream of G protein coupled receptors for agonists such as thrombin, thromboxane A(2) and ADP. The second considers the ways in which signalling events that are dependent on stable contacts between platelets can influence the state of platelet activation and thus affect thrombus growth and stability. The third focuses on the changes that are experienced by platelets as they move from their normal environment in freely-flowing plasma to a very different environment within the growing haemostatic plug, an environment in which the narrowing gaps and junctions between platelets not only facilitate communication, but also increasingly limit both the penetration of plasma and the exodus of platelet-derived bioactive molecules.

Endothelial cell specific adhesion molecule (ESAM) localizes to platelet-platelet contacts and regulates thrombus formation in vivo.

BACKGROUND: In resting platelets, endothelial cell specific adhesion molecule (ESAM) is located in alpha granules, increasing its cell surface expression following platelet activation. However, the function of ESAM on platelets is unknown. OBJECTIVE: To determine whether ESAM has a role in thrombus formation. METHODS AND RESULTS: We found that following platelet activation ESAM localizes to the junctions between adjacent platelets, suggesting a role for this protein in contact-dependent events that regulate thrombus formation. To test this hypothesis we examined the effect of ESAM deletion on platelet function. In vivo, ESAM(-/-) mice achieved more stable hemostasis than wild-type mice following tail transection, and developed larger thrombi following laser injury of cremaster muscle arterioles. In vitro, ESAM(-/-) platelets aggregated at lower concentrations of G protein-dependent agonists than wild-type platelets, and were more resistant to disaggregation. In contrast, agonist-induced calcium mobilization, alpha(IIb)beta(3) activation, alpha-granule secretion and platelet spreading, were normal in ESAM-deficient platelets. To understand the molecular mechanism by which ESAM regulates platelet activity, we utilized a PDZ domain array to identify the scaffold protein NHERF-1 as an ESAM binding protein, and further demonstrated that it associates with ESAM in both resting and activated platelets. CONCLUSIONS: These findings support a model in which ESAM localizes to platelet contacts following platelet activation in order to limit thrombus growth and stability so that the optimal hemostatic response occurs following vascular injury.

Elevated ambient glucose induces acute inflammatory events in the microvasculature: effects of insulin.

We employed intravital microscopy of the rat mesenteric microvasculature to study the effects of local hyperglycemia on leukocyte-endothelial cell interactions. Intraperitoneal injection of 6, 12.5, and 25 mmol/l D-glucose to the rat significantly and time-dependently increased leukocyte rolling and leukocyte adherence in, and leukocyte transmigration through mesenteric venules compared with control rats injected with Krebs-Henseleit (K-H) solution alone or given 25 mmol/l L-glucose intraperitoneally. The response elicited by D-glucose was associated with significant attenuation of endothelial nitric oxide (NO) release, as demonstrated by direct measurement of NO release in inferior vena caval segments isolated from rats exposed to 25 mmol/l D-glucose for 4 h (P < 0.01 vs. vena caval segments from control rats). Local application of 0.05 U/min insulin for 90 min significantly attenuated glucose-induced leukocyte rolling, adherence, and migration (P < 0.01 from 25 mmol/l D-glucose alone). Immunohistochemical localization of P-selectin expressed on endothelial surface was significantly increased 4 h after exposure of the mesenteric tissue to high ambient glucose (P < 0.01 vs. ileal venules from rats injected with K-H solution alone or 25 mmol/l L-glucose). Insulin markedly inhibited endothelial cell surface expression of P-selectin in ileal venules exposed to elevated ambient glucose in vivo (P < 0.01 vs. control rats injected with 25 mmol/l L-glucose). These data demonstrate that acute increases in ambient glucose comparable to those seen in diabetic patients are able to initiate an inflammatory response within the microcirculation. This inflammatory response to glucose is associated with upregulation of the endothelial cell adhesion molecule P-selectin and can be blocked by local application of insulin.

A new HMG-CoA reductase inhibitor, rosuvastatin, exerts anti-inflammatory effects on the microvascular endothelium: the role of mevalonic acid.

Recent studies have reported that hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase inhibitors have vasculoprotective effects independent of their lipid-lowering properties, including anti-inflammatory actions. We used intravital microscopy of the rat mesenteric microvasculature to examine the effects of rosuvastatin, a new HMG-CoA reductase inhibitor, on leukocyte-endothelium interactions induced by thrombin. Intraperitoneal administration of 0.5 and 1.25 mg kg(-1) rosuvastatin 18 h prior to the study, significantly and dose-dependently attenuated leukocyte rolling, adherence, and transmigration in the rat mesenteric microvasculature superfused with 0.5 u ml(-1) thrombin. This protective effect of rosuvastatin was reversed by intraperitoneal injection of 25 mg kg(-1) mevalonic acid 18 h before the study. Immunohistochemical detection of the endothelial cell adhesion molecule P-selectin showed a 70% decrease in endothelial cell surface expression of P-selectin in thrombin-stimulated rats given 1.25 mg kg(-1) rosuvastatin. In addition, rosuvastatin enhanced release of nitric oxide (NO) from the vascular endothelium as measured directly in rat aortic segments. Moreover, rosuvastatin failed to attenuate leukocyte-endothelium interactions in peri-intestinal venules of eNOS(-/-) mice. These data indicate that rosuvastatin exerts important anti-inflammatory effects via inhibition of endothelial cell adhesion molecule expression, and that this protective action of rosuvastatin requires release of nitric oxide by the vascular endothelium. These data also demonstrate that the mechanism of the non-lipid lowering actions of HMG-CoA reductase inhibitors in vivo may be due to reduced formation or availability of mevalonic acid within endothelial cells.

Myocardial ischemia/reperfusion injury in NADPH oxidase-deficient mice.

Previous studies have suggested that oxygen-derived free radicals are involved in the pathophysiology of myocardial ischemia/reperfusion (MI/R) injury. Specifically, neutrophils have been shown to mediate postischemic ventricular arrhythmias and myocardial necrosis. We hypothesized that MI/R injury would be reduced in the absence (-/-) of NADPH oxidase. Heterozygous control mice (n=23) and NADPH oxidase(-/-) mice (n=24) were subjected to 30 minutes of coronary artery occlusion and 24 hours of reperfusion. Myocardial area at risk per left ventricle was similar in heterozygous control hearts (55+/-3%) and NADPH oxidase(-/-) hearts (61+/-4%). Contrary to our hypothesis, the size of infarct area at risk was similar in the heterozygous control mice (42+/-4%) and NADPH oxidase(-/-) mice (34+/-5%) (P=not significant). In addition, echocardiographic examination of both groups revealed that left ventricle fractional shortening was similar in NADPH oxidase(-/-) mice (n=8; 27+/-2.5%) and heterozygous control mice (n=10; 23.3+/-3. 3%) after MI/R. Superoxide production, as detected by cytochrome c reduction, was significantly impaired (P<0.01) in NADPH oxidase(-/-) mice (n=6) compared with heterozygous mice (n=7) (0.04+/-0.03 versus 2.2+/-0.08 nmol O(2).min(-1).10(6) cells(-1)). Intravital microscopy of the inflamed mesenteric microcirculation demonstrated that leukocyte rolling and adhesion were unaffected by the absence of NADPH oxidase. Oyster glycogen-stimulated neutrophil transmigration into the peritoneum was also similar in both the heterozygous control mice and NADPH oxidase(-/-) mice (P:=not significant). These findings suggest that NADPH oxidase does not contribute to the development of myocardial injury and dysfunction after MI/R.

Use of single-primer DNA amplifications in genetic studies of peanut (Arachis hypogaea L.).

A recent approach to detecting genetic polymorphism involves the amplification of genomic DNA using single primers of arbitrary sequence. When separated electrophoretically in agarose gels, the amplification products give banding patterns that can be scored for genetic variation. The objective of this research was to apply these techniques to cultivated peanut (Arachis hypogaea L.) and related wild species to determine whether such an approach would be feasible for the construction of a genetic linkage map in peanut or for systematic studies of the genus. Two peanut cultivars, 25 unadapted germplasm lines of A. hypogaea, the wild allotetraploid progenitor of cultivated peanut (A. monticola), A. glabrata (a tetraploid species from section Rhizomatosae), and 29 diploid wild species of Arachis were evaluated for variability using primers of arbitrary sequence to amplify segments of genomic DNA. No variation in banding pattern was observed among the cultivars and germplasm lines of A. hypogaea, whereas the wild Arachis species were uniquely identified with most primers tested. Bands were scored (+/-) in the wild species and the PAUP computer program for phylogenetic analysis and the HyperRFLP program for genetic distance analysis were used to generate dendrograms showing genetic relationships among the diploid Arachis species evaluated. The two analyses produced nearly identical dendrograms of species relationships. In addition, approximately 100 F2 progeny from each of two interspecific crosses were evaluated for segregation of banding patterns. Although normal segregation was observed among the F2 progeny from both crosses, banding patterns were quite complex and undesirable for use in genetic mapping. The dominant behavior of the markers prevented the differentiation of heterozygotes from homozygotes with certainty, limiting the usefulness of arbitrary primer amplification products as markers in the construction of a genetic linkage map in peanut.