A systems approach to hemostasis: 4. How hemostatic thrombi limit the loss of plasma-borne molecules from the microvasculature.

Previous studies have shown that hemostatic thrombi formed in response to penetrating injuries have a core of densely packed, fibrin-associated platelets overlaid by a shell of less-activated, loosely packed platelets. Here we asked, first, how the diverse elements of this structure combine to stem the loss of plasma-borne molecules and, second, whether antiplatelet agents and anticoagulants that perturb thrombus structure affect the re-establishment of a tight vascular seal. The studies combined high-resolution intravital microscopy with a photo-activatable fluorescent albumin marker to simultaneously track thrombus formation and protein transport following injuries to mouse cremaster muscle venules. The results show that protein loss persists after red cell loss has ceased. Blocking platelet deposition with an αIIbß3antagonist delays vessel sealing and increases extravascular protein accumulation, as does either inhibiting adenosine 5'-diphosphate (ADP) P2Y12receptors or reducing integrin-dependent signaling and retraction. In contrast, sealing was unaffected by introducing hirudin to block fibrin accumulation or a Gi2α gain-of-function mutation to expand the thrombus shell. Collectively, these observations describe a novel approach for studying vessel sealing after injury in real time in vivo and show that (1) the core/shell architecture previously observed in arterioles also occurs in venules, (2) plasma leakage persists well beyond red cell escape and mature thrombus formation, (3) the most critical events for limiting plasma extravasation are the stable accumulation of platelets, ADP-dependent signaling, and the emergence of a densely packed core, not the accumulation of fibrin, and (4) drugs that affect platelet accumulation and packing can delay vessel sealing, permitting protein escape to continue.

In microfluidico: Recreating in vivo hemodynamics using miniaturized devices.

Microfluidic devices create precisely controlled reactive blood flows and typically involve: (i) validated anticoagulation/pharmacology protocols, (ii) defined reactive surfaces, (iii) defined flow-transport regimes, and (iv) optical imaging. An 8-channel device can be run at constant flow rate or constant pressure drop for blood perfusion over a patterned collagen, collagen/kaolin, or collagen/tissue factor (TF) to measure platelet, thrombin, and fibrin dynamics during clot growth. A membrane-flow device delivers a constant flux of platelet agonists or coagulation enzymes into flowing blood. A trifurcated device sheaths a central blood flow on both sides with buffer, an ideal approach for on-chip recalcification of citrated blood or drug delivery. A side-view device allows clotting on a porous collagen/TF plug at constant pressure differential across the developing clot. The core-shell architecture of clots made in mouse models can be replicated in this device using human blood. For pathological flows, a stenosis device achieves shear rates of >100,000 s(-1) to drive plasma von Willebrand factor (VWF) to form thick long fibers on collagen. Similarly, a micropost-impingement device creates extreme elongational and shear flows for VWF fiber formation without collagen. Overall, microfluidics are ideal for studies of clotting, bleeding, fibrin polymerization/fibrinolysis, cell/clot mechanics, adhesion, mechanobiology, and reaction-transport dynamics.

Microfluidic assessment of functional culture-derived platelets in human thrombi under flow.

Despite their clinical significance, human platelets are not amenable to genetic manipulation, thus forcing a reliance on mouse models. Culture-derived platelets (CDPs) from human peripheral blood CD34(+) cells can be genetically altered and may eventually be used for transfusions. By use of microfluidics, the time-dependent incorporation of CD41(+)CD42(+) CDPs into clots was measured using only 54,000 CDPs doped into 27 µL of human whole blood perfused over collagen at a wall shear rate of 100 sec(-1). With the use of fluorescence-labeled human platelets (instead of CDPs) doped between 0.25% and 2% of total platelets, incorporation was highly quantitative and allowed monitoring of the anti-αIIbß3 antagonism that occurred after collagen adhesion. CDPs were only 15% as efficient as human platelets in their incorporation into human thrombi under flow, although both cell types were equally antagonized by αIIbß3 inhibition. Transient transfection allowed the monitoring of GFP(+) human CDP incorporation into clots. This assay quantifies genetically altered CDP function under flow.

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 (ΔP) 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 ΔP, we found thrombin to be highly localized in the P-selectin(+) core of hemostatic clots. Increasing ΔP 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-γ'-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 ΔP. Also, γ'-fibrinogen had a role in venous but not arterial conditions.

Rapid on-chip recalcification and drug dosing of citrated whole blood using microfluidic buffer sheath flow.

Millions of clotting tests each year require recalcification of blood treated with sodium citrate, a calcium chelator that prevents prothrombinase assembly. We validated a converging trifurcated microfluidic device to measure platelet and fibrin accumulation following on-chip recalcification of citrated whole blood. Recalcification was accomplished by sheathing the blood with Ca2+ buffer. Fluorescein rapidly diffused across the buffer-blood interface (achieving 62.5% of maximum centerline concentration within ~4 cm of flow), while albumin remained relatively unchanged in blood due to its lower diffusivity (<20% decrease). Since Ca2+ diffuses faster than fluorescein, full recalcification of whole blood was achieved within ~1 cm of flow prior to encountering a collagen/tissue surface. Platelet and fibrin were reduced by 87.3% and 99.0%, respectively, when the sheath buffer was Ca2+-free. A 30-min preincubation of citrated whole blood prior to on-chip recalcification increased platelet (159%) and fibrin (86.6%) deposition, compared to 5-min preincubation, likely due to factor XIIa generation in citrated blood. The P2Y1 inhibitor, MRS-2179, was delivered by diffusion into flowing blood and inhibited platelet deposition on collagen with a calculated IC50 of 0.155 µM. On-chip recalcification and drug dosing of citrated blood allows for assays of platelet function in a whole blood milieu under flow.

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.

Side view thrombosis microfluidic device with controllable wall shear rate and transthrombus pressure gradient.

Hemodynamic conditions vary throughout the vasculature, creating diverse environments in which platelets must respond. To stop bleeding, a growing platelet deposit must be assembled in the presence of fluid wall shear stress (τw) and a transthrombus pressure gradient (ΔP) that drives bleeding. We designed a microfluidic device capable of pulsing a fluorescent solute through a developing thrombus forming on collagen ± tissue factor (TF), while independently controlling ΔP and τw. Computer control allowed step changes in ΔP with a rapid response time of 0.26 mm Hg s(-1) at either venous (5.2 dynes cm(-2)) or arterial (33.9 dynes cm(-2)) wall shear stresses. Side view visualization of thrombosis with transthrombus permeation allowed for quantification of clot structure, height, and composition at various ΔP. Clot height was reduced 20% on collagen/TF and 28% on collagen alone when ΔP was increased from 20.8 to 23.4 mm Hg at constant arterial shear stress. When visualized with a platelet-targeting thrombin sensor, intrathrombus thrombin levels decreased by 62% as ΔP was increased from 0 to 23.4 mm Hg across the thrombus-collagen/TF barrier, consistent with convective removal of thrombogenic solutes due to pressure-driven permeation. Independent of ΔP, the platelet deposit on collagen had a permeability of 5.45 × 10(-14) cm(2), while the platelet/fibrin thrombus on collagen/TF had a permeability of 2.71 × 10(-14) cm(2) (comparable to that of an intact endothelium). This microfluidic design allows investigation of the coupled processes of platelet deposition and thrombin/fibrin generation in the presence of controlled transthrombus permeation and wall shear stress.

Blood clots are rapidly assembled hemodynamic sensors: flow arrest triggers intraluminal thrombus contraction.

OBJECTIVE: Blood clots form under flow during intravascular thrombosis or vessel leakage. Prevailing hemodynamics influence thrombus structure and may regulate contraction processes. A microfluidic device capable of flowing human blood over a side channel plugged with collagen (± tissue factor) was used to measure thrombus permeability (κ) and contraction at controlled transthrombus pressure drops. METHODS AND RESULTS: The collagen (κ(collagen)=1.98 × 10(-11) cm(2)) supported formation of a 20-µm thick platelet layer, which unexpectedly underwent massive platelet retraction on flow arrest. This contraction resulted in a 5.34-fold increase in permeability because of collagen restructuring. Without stopping flow, platelet deposits (no fibrin) had a permeability of κ(platelet)=5.45 × 10(-14) cm(2) and platelet-fibrin thrombi had κ(thrombus)=2.71 × 10(-14) cm(2) for ΔP=20.7 to 23.4 mm Hg, the first ever measurements for clots formed under arterial flow (1130 s(-1) wall shear rate). Platelet sensing of flow cessation triggered a 4.6- to 6.5-fold (n=3, P<0.05) increase in contraction rate, which was also observed in a rigid, impermeable parallel-plate microfluidic device. This triggered contraction was blocked by the myosin IIA inhibitor blebbistatin and by inhibitors of thromboxane A2 (TXA(2)) and ADP signaling. In addition, flow arrest triggered platelet intracellular calcium mobilization, which was blocked by TXA(2)/ADP inhibitors. As clots become occlusive or blood pools following vessel leakage, the flow diminishes, consequently allowing full platelet retraction. CONCLUSIONS: Flow dilution of ADP and thromboxane regulates platelet contractility with prevailing hemodynamics, a newly defined flow-sensing mechanism to regulate clot function.

Thrombus growth and embolism on tissue factor-bearing collagen surfaces under flow: role of thrombin with and without fibrin.

OBJECTIVE: At sites of vascular injury, thrombin is an important mediator in thrombus growth and stability. Using microfluidic flow devices as well as patterned surfaces of collagen and tissue factor (TF), we sought to determine the role that fibrin plays in clot stability without interfering with the production of thrombin. METHODS AND RESULTS: We deployed an 8-channel microfluidic device to study coagulation during corn trypsin inhibitor-treated (XIIa-inhibited) whole blood perfusion over lipidated TF linked to a fibrillar collagen type 1 surface. Clot growth and embolization were measured at initial inlet venous (200 s(-1)) or arterial (1000 s(-1)) wall shear rates under constant flow rate or pressure relief mode in the presence or absence of Gly-Pro-Arg-Pro (GPRP) to block fibrin polymerization. Numerical calculations for each mode defined hemodynamic forces on the growing thrombi. In either mode at inlet venous flow, increasing amounts of TF on the surface led to a modest dose-dependent increase (up to 2-fold) in platelet deposition, but resulted in massive fibrin accumulation (>50-fold) only when exceeding a critical TF threshold. At a venous inlet flow, GPRP led to a slight 20% increase in platelet accumulation (P<0.01) in pressure relief mode with thrombi resisting ≈1500 s(-1) before full channel occlusion. GPRP-treated thrombi were unstable under constant flow rate, where shear forces caused embolization at a maximum shear rate of ≈2300 s(-1) (69 dynes/cm2). In constant flow rate mode, the nonocclusive platelet-fibrin deposits (no GPRP) withstood maximum shear rates of ≈29 000 s(-1) (870 dyne/cm2) at ≈95% of full channel occlusion. For arterial inlet shear rate, embolization was marked for either mode with GPRP present when shear forces reached 87 dynes/cm2 (≈2900 s(-1)). Under constant flow rate, platelet-fibrin deposits (no GPRP) withstood maximums of 2400 dynes/cm2 (80,000 s(-1)) at ≈90% of full channel occlusion prior to embolization. CONCLUSIONS: Fibrin increased clot strength by 12- to 28-fold. Under pressure relief mode, ≈2-fold more fibrin was produced under venous flow (P<0.001). These studies define embolization criteria for clots formed with surface TF-triggered thrombin production (±fibrin) under venous and arterial flows.