Contact Pathway Function During Human Whole Blood Clotting on Procoagulant Surfaces.

Microfluidic thrombosis assays allow the control of anticoagulation, hemodynamics, pharmacology, and procoagulant surfaces containing collagen ± tissue factor (TF). With corn trypsin inhibitor (CTI) ranging from low (1-4 µg/mL) to high levels (40-60 µg/mL), the function of Factor XIIa (FXIIa) can be modulated in the presence of low or high surface TF. With high CTI and no collagen/TF in the assay, no thrombin is generated during 15-min microfluidic perfusion. At low CTI (no TF), the generation of FXIa leads to fibrin polymerization at ~300 s after the initiation of flow over collagen, an onset time shortened at zero CTI and prolonged at high CTI. The engagement of FXIa was difficult to observe for clotting on high TF surfaces due to the dominance of the extrinsic pathway. Low TF surfaces allowed observable crosstalk between extrinsic pathway generation of thrombin and thrombin-mediated activation of FXIa, a feedback detected at >5 min and attenuated with polyphosphate inhibitor. From thrombin-antithrombin immunoassay of the effluent of blood flowing over collagen/TF, the majority of thrombin was found captured on intrathrombus fibrin. Additionally, extreme shear rates (>10,000 s-1) can generate massive von Willebrand Factor fibers that capture FXIIa and FXIa to drive fibrin generation, an event that facilitates VWF fiber dissolution under fibrinolytic conditions. Finally, we found that occlusive sterile thrombi subjected to pressure drops >70 mm-Hg/mm-clots have interstitial stresses sufficient to drive NETosis. These microfluidic studies highlight the interaction of contact pathway factors with the extrinsic pathway, platelet polyphosphate, VWF fibers, and potentially shear-induced NETs.

Using microfluidic devices to study thrombosis in pathological blood flows.

Extreme flows can exist within pathological vessel geometries or mechanical assist devices which create complex forces and lead to thrombogenic problems associated with disease. Turbulence and boundary layer separation are difficult to obtain in microfluidics due to the low Reynolds number flow in small channels. However, elongational flows, extreme shear rates and stresses, and stagnation point flows are possible using microfluidics and small perfusion volumes. In this review, a series of microfluidic devices used to study pathological blood flows are described. In an extreme stenosis channel pre-coated with fibrillar collagen that rapidly narrows from 500 µm to 15 µm, the plasma von Willebrand Factor (VWF) will elongate and assemble into thick fiber bundles on the collagen. Using a micropost-impingement device, plasma flow impinging on the micropost generates strong elongational and wall shear stresses that trigger the growth of a VWF bundle around the post (no collagen required). Using a stagnation-point device to mimic the zone near flow reattachment, blood can be directly impinged upon a procoagulant surface of collagen and the tissue factor. Clots formed at the stagnation point of flow impingement have a classic core-shell architecture where the core is highly activated (P-selectin positive platelets and fibrin rich). Finally, within occlusive clots that fill a microchannel, the Darcy flow driven by ΔP/L > 70 mm-Hg/mm-clot is sufficient to drive NETosis of entrapped neutrophils, an event not requiring either thrombin or fibrin. Novel microfluidic devices are powerful tools to access physical environments that exist in human disease.

Potent Thrombolytic Effect of N-Acetylcysteine on Arterial Thrombi.

BACKGROUND: Platelet cross-linking during arterial thrombosis involves von Willebrand Factor (VWF) multimers. Therefore, proteolysis of VWF appears promising to disaggregate platelet-rich thrombi and restore vessel patency in acute thrombotic disorders such as ischemic stroke, acute coronary syndrome, or acute limb ischemia. N-Acetylcysteine (NAC, a clinically approved mucolytic drug) can reduce intrachain disulfide bonds in large polymeric proteins. In the present study, we postulated that NAC might cleave the VWF multimers inside occlusive thrombi, thereby leading to their dissolution and arterial recanalization. METHODS: Experimental models of thrombotic stroke induced by either intra-arterial thrombin injection or ferric chloride application followed by measurement of cerebral blood flow using a combination of laser Doppler flowmetry and MRI were performed to uncover the effects of NAC on arterial thrombi. To investigate the effect of NAC on larger vessels, we also performed ferric chloride-induced carotid artery thrombosis. In vitro experiments were performed to study the molecular bases of NAC thrombolytic effect, including platelet aggregometry, platelet-rich thrombi lysis assays, thromboelastography (ROTEM), and high-shear VWF string formation using microfluidic devices. We also investigated the putative prohemorrhagic effect of NAC in a mouse model of intracranial hemorrhage induced by in situ collagenase type VII injection. RESULTS: We demonstrated that intravenous NAC administration promotes lysis of arterial thrombi that are resistant to conventional approaches such as recombinant tissue-type plasminogen activator, direct thrombin inhibitors, and antiplatelet treatments. Through in vitro and in vivo experiments, we provide evidence that the molecular target underlying the thrombolytic effects of NAC is principally the VWF that cross-link platelets in arterial thrombi. Coadministration of NAC and a nonpeptidic GpIIb/IIIa inhibitor further improved its thrombolytic efficacy, essentially by accelerating thrombus dissolution and preventing rethrombosis. Thus, in a new large-vessel thromboembolic stroke model in mice, this cotreatment significantly improved ischemic lesion size and neurological outcome. It is important to note that NAC did not worsen hemorrhagic stroke outcome, suggesting that it exerts thrombolytic effects without significantly impairing normal hemostasis. CONCLUSIONS: We provide evidence that NAC is an effective and safe alternative to currently available antithrombotic agents to restore vessel patency after arterial occlusion.

Thrombi produced in stagnation point flows have a core-shell structure.

INTRODUCTION: In regions of flow separation/reattachment within diseased arteries, the local hemodynamics can result in stagnation point flow that provides an atypical environment in atherosclerosis. Impinging flows occur with recirculation eddies distal of coronary stenosis or diseased carotid bifurcations. METHODS: By perfusing whole blood directly perpendicular to a fibrillar collagen thrombotic surface, a microfluidic device produced a stagnation point flow. Side view visualization of thrombosis in this assay allowed for observation of clot structure and composition at various flow rates and blood biochemistry conditions. RESULTS: For clotting over collagen/tissue factor surfaces, platelet thrombi formed in this device displayed a core-shell architecture with a fibrin-rich, platelet P-selectin-positive core and an outer platelet P-selectin-negative shell. VWF was detected in clots at low and high shear, but when N-acetylcysteine was added to the whole blood, both platelet and VWF deposition were markedly decreased at either low or high flow. To further examine the source of clot stability, 1 mM GPRP was added to prevent fibrin formation while allowing the PAR1/4-cleaving activity of thrombin to progress. The inhibition of fibrin polymerization did not change the overall structure of the clots, demonstrating the stability of these clots without fibrin. CONCLUSION: Impinging flow microfluidics generate thrombi with a core-shell structure.

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.