John D. Welsh

A systems approach to hemostasis: 3. Thrombus consolidation regulates intrathrombus solute transport and local thrombin activity

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.

A systems approach to hemostasis: 1. The interdependence of thrombus architecture and agonist movements in the gaps between platelets.

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.

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

Summary.  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.

A systems approach to hemostasis: 2. Computational analysis of molecular transport in the thrombus microenvironment

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 less-activated, 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.

Shaping the platelet response to vascular injury

Purpose of reviewSeveral decades of work by many investigators have elucidated the major signaling pathways responsible for platelet activation. Still to be fully understood is how these pathways are integrated into a single network and how changing conditions within a growing thrombus affect that network. In this review we will consider some of the recent studies that address these issues and describe a model that provides insights into platelet activation as it occurs in vivo. Recent findingsGenetic and pharmacologic studies performed in vivo have demonstrated that platelet activation during hemostasis and thrombosis is heterogeneous. Those studies indicate that distinct platelet activation pathways are not merely redundant, but are coordinated in time and space to achieve an optimal response. This coordination is achieved at least in part by the evolving distribution of platelet agonists and changes in solute transport within a hemostatic plug. SummaryStudies examining the coordination of platelet signaling in time and space continue to increase our understanding of hemostasis and thrombosis. In addition to helping to decipher platelet biology, the results have implications for the understanding of new and existing antiplatelet agents and their potential risks.

Fibrin, γ′-Fibrinogen, and Transclot Pressure Gradient Control Hemostatic Clot Growth During Human Blood Flow Over a Collagen/Tissue Factor Wound

Objective— Biological and physical factors interact to modulate blood response in a wounded vessel, resulting in a hemostatic clot or an occlusive thrombus. Flow and pressure differential (&Dgr;P) across the wound from the lumen to the extravascular compartment may impact hemostasis and the observed core/shell architecture. We examined physical and biological factors responsible for regulating thrombin-mediated clot growth. Approach and Results— Using factor XIIa-inhibited human whole blood perfused in a microfluidic device over collagen/tissue factor at controlled wall shear rate and &Dgr;P, we found thrombin to be highly localized in the P-selectin+ core of hemostatic clots. Increasing &Dgr;P from 9 to 29 mm Hg (wall shear rate=400 s−1) reduced P-selectin+ core size and total clot size because of enhanced extravasation of thrombin. Blockade of fibrin polymerization with 5 mmol/L Gly-Pro-Arg-Pro dysregulated hemostasis by enhancing both P-selectin+ core size and clot size at 400 s−1 (20 mm Hg). For whole-blood flow (no Gly-Pro-Arg-Pro), the thickness of the P-selectin-negative shell was reduced under arterial conditions (2000 s−1, 20 mm Hg). Consistent with the antithrombin-1 activity of fibrin implicated with Gly-Pro-Arg-Pro, anti-&ggr;′-fibrinogen antibody enhanced core-localized thrombin, core size, and overall clot size, especially at venous (100 s−1) but not arterial wall shear rates (2000 s−1). Pathological shear (15 000 s−1) and Gly-Pro-Arg-Pro synergized to exacerbate clot growth. Conclusions— Hemostatic clotting was dependent on core-localized thrombin that (1) triggered platelet P-selectin display and (2) was highly regulated by fibrin and the transclot &Dgr;P. Also, &ggr;′-fibrinogen had a role in venous but not arterial conditions.

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.

Platelet‐targeting thiol reduction sensor detects thiol isomerase activity on activated platelets in mouse and human blood under flow

Essentials Protein disulfide isomerases may have an essential role in thrombus formation. A platelet‐binding sensor (PDI‐sAb) was developed to detect thiol reductase activity under flow. Primary human platelet adhesion to collagen at 200 s−1 was correlated with the PDI‐sAb signal. Detected thiol reductase activity was localized in the core of growing thrombi at the site of injury in mice.

Neutrophil accumulation and NET release contribute to thrombosis in HIT

Heparin-induced thrombocytopenia (HIT) is an immune-mediated thrombocytopenic disorder associated with a severe prothrombotic state. We investigated whether neutrophils and neutrophil extracellular traps (NETs) contribute to the development of thrombosis in HIT. Using an endothelialized microfluidic system and a murine passive immunization model, we show that HIT induction leads to increased neutrophil adherence to venous endothelium. In HIT mice, endothelial adherence is enhanced immediately downstream of nascent venous thrombi, after which neutrophils undergo retrograde migration via a CXCR2-dependent mechanism to accumulate into the thrombi. Using a microfluidic system, we found that PF4 binds to NETs, leading them to become compact and DNase resistant. PF4-NET complexes selectively bind HIT antibodies, which further protect them from nuclease digestion. In HIT mice, inhibition of NET formation through Padi4 gene disruption or DNase treatment limited venous thrombus size. PAD4 inactivation did affect arterial thrombi or severity of thrombocytopenia in HIT. Thus, neutrophil activation contributes to the development of venous thrombosis in HIT by enhancing neutrophil-endothelial adhesion and neutrophil clot infiltration, where incorporated PF4-NET-HIT antibody complexes lead to thrombosis propagation. Inhibition of neutrophil endothelial adhesion, prevention of neutrophil chemokine-dependent recruitment of neutrophils to thrombi, or suppression of NET release should be explored as strategies to prevent venous thrombosis in HIT.