Thomas V. Colace

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.

Multiscale prediction of patient-specific platelet function under flow

During thrombotic or hemostatic episodes, platelets bind collagen and release ADP and thromboxane A(2), recruiting additional platelets to a growing deposit that distorts the flow field. Prediction of clotting function under hemodynamic conditions for a patients platelet phenotype remains a challenge. A platelet signaling phenotype was obtained for 3 healthy donors using pairwise agonist scanning, in which calcium dye-loaded platelets were exposed to pairwise combinations of ADP, U46619, and convulxin to activate the P2Y(1)/P2Y(12), TP, and GPVI receptors, respectively, with and without the prostacyclin receptor agonist iloprost. A neural network model was trained on each donors pairwise agonist scanning experiment and then embedded into a multiscale Monte Carlo simulation of donor-specific platelet deposition under flow. The simulations were compared directly with microfluidic experiments of whole blood flowing over collagen at 200 and 1000/s wall shear rate. The simulations predicted the ranked order of drug sensitivity for indomethacin, aspirin, MRS-2179 (a P2Y(1) inhibitor), and iloprost. Consistent with measurement and simulation, one donor displayed larger clots and another presented with indomethacin resistance (revealing a novel heterozygote TP-V241G mutation). In silico representations of a subjects platelet phenotype allowed prediction of blood function under flow, essential for identifying patient-specific risks, drug responses, and novel genotypes.

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.

Microfluidics and Coagulation Biology

The study of blood ex vivo can occur in closed or open systems, with or without flow. Microfluidic devices, which constrain fluids to a small (typically submillimeter) scale, facilitate analysis of platelet function, coagulation biology, cellular biorheology, adhesion dynamics, and pharmacology and, as a result, can be an invaluable tool for clinical diagnostics. An experimental session can accommodate hundreds to thousands of unique clotting, or thrombotic, events. Using microfluidics, thrombotic events can be studied on defined surfaces of biopolymers, matrix proteins, and tissue factor, under constant flow rate or constant pressure drop conditions. Distinct shear rates can be generated on a device using a single perfusion pump. Microfluidics facilitated both the determination of intraluminal thrombus permeability and the discovery that platelet contractility can be activated by a sudden decrease in flow. Microfluidic devices are ideal for multicolor imaging of platelets, fibrin, and phosphatidylserine and provide a human blood analog to mouse injury models. Overall, microfluidic advances offer many opportunities for research, drug testing under relevant hemodynamic conditions, and clinical diagnostics.

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. Conclusion—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.

Direct Observation of von Willebrand Factor Elongation and Fiber Formation on Collagen During Acute Whole Blood Exposure to Pathological Flow

Objective—In severe stenosis, von Willebrand Factor (vWF) experiences millisecond exposures to pathological wall shear rates (&ggr;w). We sought to evaluate the deposition of vWF onto collagen surfaces under flow in these environments. Methods and Results—Distinct from viscometry experiments that last many seconds, we deployed microfluidic devices for single-pass perfusion of whole blood or platelet-free plasma over fibrillar type 1 collagen (<50 ms transit time) at pathological &ggr;w or spatial wall shear rate gradients (grad &ggr;w). Using fluorescent anti-vWF, long thick vWF fibers (>20 &mgr;m) bound to collagen were visualized at constant &ggr;w>30000 s–1 during perfusion of platelet-free plasma, a process enhanced by EDTA. Rapid acceleration or deceleration of EDTA platelet-free plasma at grad &ggr;w=±1.1×105 to ±4.3×107 s–1/cm did not promote vWF deposition. At 19400 s–1, EDTA blood perfusion resulted in rolling vWF–platelet nets, although blood perfusion (normal Ca2+) generated large vWF/platelet deposits that repeatedly embolized and were blocked by anti-glycoprotein Ib or the &agr;IIb&bgr;3 inhibitor GR144053 and did not require grad &ggr;w. Blood perfusion at venous shear rate (200 s–1) produced a stable platelet deposit that was a substrate for massive but unstable vWF–platelet aggregates when flow was increased to 7800 s–1. Conclusion—Triggered by collagen and enhanced by platelet glycoprotein Ib and &agr;IIb&bgr;3, vWF fiber formation occurred during acute exposures to pathological &ggr;w and did not require gradients in wall shear rate.

Relipidated tissue factor linked to collagen surfaces potentiates platelet adhesion and fibrin formation in a microfluidic model of vessel injury

Microfluidic devices allow for the controlled perfusion of human or mouse blood over defined prothrombotic surfaces at venous and arterial shear rates. To mimic in vivo injuries such a plaque rupture, the need exists to link lipidated tissue factor (TF) to surface-bound collagen fibers. Recombinant TF was relipidated in liposomes of phosphatidylserine/phosphatidylcholine/biotin-linked phosphatidylethanolamine (20:79:1 PS/PC/bPE molar ratio). Collagen was patterned in a 250-μm-wide stripe and labeled with biotinylated anticollagen antibody which was then bound with streptavidin, allowing the subsequent capture of the TF liposomes. To verify and detect the TF liposome-collagen assembly, individual molecular complexes of TF-factor VIIa on collagen were visualized using the proximity ligation assay (PLA) to produce discretely localized fluorescent events that were strictly dependent on the presence of factor VIIa and primary antibodies against TF or factor VIIa. Perfusion for 450 s (wall shear rate, 200 s(-1)) of corn trypsin inhibitor (CTI, a factor XIIa inhibitor) treated whole blood over the stripe of TF-collagen enhanced platelet adhesion by 30 ± 8% (p < 0.001) and produced measurable fibrin (>50-fold increase) as compared to surfaces lacking TF. PS/PC/bPE liposomes lacking TF resulted in no enhancement of platelet deposition. Essentially no fibrin was formed during perfusion over collagen surfaces or collagen surfaces with liposomes lacking TF despite the robust platelet deposition, indicating a lack of kinetically significant platelet-borne tissue factor in healthy donor blood. This study demonstrates a reliable approach to link functionally active TF to collagen for microfluidic thrombosis studies.

Microfluidic assay of hemophilic blood clotting: Distinct deficits in platelet and fibrin deposition at low factor levels

Coagulation factor deficiencies create a range of bleeding phenotypes. Microfluidic devices offer controlled hemodynamics and defined procoagulant triggers for measurement of clotting under flow.

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.