Recombinant factor VIIa addition to haemophilic blood perfused over collagen/tissue factor can sufficiently bypass the factor IXa/VIIIa defect to rescue fibrin generation.

INTRODUCTION: Factor VIII (FVIII) or factor IX (FIX)-deficient haemophilic patients display deficits in platelet and fibrin deposition under flow detectable in microfluidics. Compared to fibrin generation, decreased platelet deposition in haemophilic blood flow is more easily rescued with recombinant factor VIIa (rFVIIa), whereas rFVIIa requires FXIIa participation to generate fibrin when tissue factor (TF) is absent. AIMS: Perfusion of haemophilic whole blood (WB) over collagen/TF surfaces was used to determine whether rFVIIa/TF was sufficient to bypass poor FIXa/FVIIIa function in blood from patients with haemophilia A and B. METHODS: Whole blood treated with high-dose corn trypsin inhibitor (40 µg mL-1 ) from seven healthy donors and 10 patients was perfused over fibrillar collagen presenting low or high TF (TFlow or TFhigh ) at wall shear rate of 100 s-1 . RESULTS: With WB from healthy controls, platelet deposition and fibrin accumulation increased as TF increased. Factor-deficient WB (1-3% of normal) displayed striking deficits in platelet deposition and fibrin formation at either TFlow or TFhigh . In contrast, mildly factor-deficient WB (14-32%) supported fibrin formation under flow on TFhigh /collagen. With either TFlow or TFhigh , exogenously added rFVIIa (20 nm) increased platelet deposition and fibrin accumulation in WB from factor-deficient patients (1-3% of normal) to levels commensurate with untreated healthy WB. CONCLUSION: The absence of FIXa/FVIIIa in patients with severe haemophilia results in deficits in fibrin formation that cannot be rescued by wall-derived TF ex vivo. The effects of rFVIIa on platelet adhesion and rFVIIa/TF can act together to reinforce thrombin generation, platelet deposition and fibrin formation under flow.

Recombinant factor VIIa enhances platelet deposition from flowing haemophilic blood but requires the contact pathway to promote fibrin deposition.

In prior microfluidic studies with haemophilic blood perfused over collagen, we found that a severe deficiency (<1% factor level) reduced platelet and fibrin deposition, while a moderate deficiency (1-5%) only reduced fibrin deposition. We investigated: (i) the differential effect of rFVIIa (0.04-20 nm) on platelet and fibrin deposition, and (ii) the contribution of the contact pathway to rFVIIa-induced haemophilic blood clotting. Haemophilic or healthy blood with low and high corn trypsin inhibitor (CTI, 4 or 40 µg mL(-1) ) was perfused over collagen at an initial venous wall shear rate of 100 s(-1) . At 100 s(-1) wall shear rate, where FXIIa leads to thrombin production without added tissue factor, FXI-deficient blood (3%) or severely FVIII-deficient blood (<1%) produced no fibrin at either CTI level. Whereas rFVIIa potently enhanced platelet deposition, fibrin generation was not rescued. Distinct from the high CTI condition, engagement of the contact pathway (low CTI) in moderately FVIII-deficient (3%) or moderately FIX-deficient blood (5%) resulted in enhanced platelet and fibrin deposition following 4 nm rFVIIa supplementation. In mildly FVIII-deficient blood (15%) at <24 h since haemostatic therapy, rFVIIa enhanced both platelet and fibrin generation in either CTI condition although fibrin was produced more quickly and abundantly in low CTI. For tissue factor-free conditions of severe haemophilic blood clotting, we conclude that rFVIIa reliably generates low levels of 'signaling' thrombin sufficient to enhance platelet deposition on collagen, but is insufficient to drive fibrin polymerization unless potentiated by the contact pathway.

Activation of Most Toll-Like Receptors in Whole Human Blood Attenuates Platelet Deposition on Collagen under Flow.

Platelets have toll-like receptors (TLRs); however, their function in thrombosis or hemostasis under flow conditions is not fully known. Thrombin-inhibited anticoagulated whole blood was treated with various TLR agonists and then perfused over fibrillar collagen using microfluidic assay at venous wall shear rate (100 s-1). Platelet deposition was imaged with fluorescent anti-CD61. For perfusion of whole blood without TLR agonist addition, platelets rapidly accumulated on collagen and eventually occluded the microchannels. Interestingly, most of the tested TLR agonists (Pam3CKS4, MALP-2, polyinosinic-polycytidylic acid HMW, imiquimod, and CpG oligodeoxynucleotides) strongly reduced platelet deposition on collagen, while only the TLR4 agonist endotoxin lipopolysaccharide (LPS) enhanced deposition. Following 90 sec of deposition under flow of untreated blood, the addition of various TLR-7 agonists (imiquimod, vesatolimod, and GSK2245035) all caused immediate blockade of further platelet deposition. Since TLR signaling can activate nuclear factor-kappaB (NF-κB), the IKK-inhibitor (IKK inhibitor VII) and NF-κB inhibitor (Bay 11-7082) were tested. The IKK/NF-κB inhibitors strongly inhibited platelet deposition under flow. Furthermore, addition of Pam3CSK4 (TLR1/2 ligand), MALP-2 (TLR2/6 ligand), and Imquimod (TLR7 ligand) reduced phosphotidylserine (PS) exposure. Activation of TLR1/2, TLR2/6, TLR3, TLR7, and TLR9 in whole blood reduced platelet deposition under flow on collagen; however, LPS (major Gram negative bacterial pathogenic component) activation of LTR4 was clearly prothrombotic.

Anti-GPVI Fab reveals distinct roles for GPVI signaling in the first platelet layer and subsequent layers during microfluidic clotting on collagen with or without tissue factor.

The collagen receptor glycoprotein VI (GPVI) drives strong platelet activation, however its role at later stages of clotting remains less clear. Controlled timing of addition of anti-human GPVI Fab (clone E12) with microfluidic venous whole blood flow over collagen (± lipidated tissue factor, TF) produced distinct effects on platelets, fibrin, P-selectin exposure, and phosphatidylserine (PS) exposure. On collagen alone, Fab present initially potently reduced platelet deposition on collagen, while Fab added 90 s after initial platelet deposition, stopped subsequent platelet accumulation (despite the absence of fibrin). With thrombin generation via TF, Fab added at either t = 0 or 90 s had no effect on platelet deposition. However, Fab added initially, but not at 90-s, blocked fibrin formation. Gly-Pro-Arg-Pro ablated fibrin formation without effect on platelet accumulation (regardless of Fab added at t = 0 or 90 s), indicating thrombin signaling can suffice over GPVI signaling. Still, Fab moderately reduced P-selectin exposure with thrombin present and fibrin absent. On collagen/TF, Fab present initially ablated PS exposure, but had no effect when added 30 to 90-s later. The thrombin generated via PS exposure had an important role in driving platelet deposition in the presence of Fab, since inhibition of PS via annexin V binding in the presence of Fab significantly inhibited platelet deposition. We conclude GPVI signaling in the first platelet layer on collagen dictates thrombin and fibrin production, but the role of GPVI at subsequent times after formation of the first monolayer is obscured by thrombin-induced signaling.

Thermal shift assay to probe melting of thrombin, fibrinogen, fibrin monomer, and fibrin: Gly-Pro-Arg-Pro induces a fibrin monomer-like state in fibrinogen.

BACKGROUND: Thrombin activates fibrinogen and binds the fibrin E-domain (Kd ~ 2.8 µM) and the splice variant γ'-domain (Kd ~ 0.1 µM). We investigated if the loading of D-Phe-Pro-Arg-chloromethylketone inhibited thrombin (PPACK-thrombin) onto fibrin could enhance fibrin stability. METHODS: A 384-well plate thermal shift assay (TSA) with SYPRO-orange provided melting temperatures (Tm) of thrombin, PPACK-thrombin, fibrinogen, fibrin monomer, and fibrin. RESULTS: Large increases in Tm indicated that calcium led to protein stabilization (0 vs. 2 mM Ca2+) for fibrinogen (54.0 vs. 62.3 °C) and fibrin (62.3 vs. 72.2 °C). Additionally, active site inhibition with PPACK dramatically increased the Tm of thrombin (58.3 vs. 78.3 °C). Treatment of fibrinogen with fibrin polymerization inhibitor GPRP increased fibrinogen stability by ΔTm = 9.3 °C, similar to the ΔTm when fibrinogen was converted to fibrin monomer (ΔTm = 8.8 °C) or to fibrin (ΔTm = 10.4 °C). Addition of PPACK-thrombin at high 5:1 M ratio to fibrin(ogen) had little effect on fibrin(ogen) Tm values, indicating that thrombin binding does not detectably stabilize fibrin via a putative bivalent E-domain to γ'-domain interaction. CONCLUSIONS: TSA was a sensitive assay of protein stability and detected: (1) the effects of calcium-stabilization, (2) thrombin active site labeling, (3) fibrinogen conversion to fibrin, and (4) GPRP induced changes in fibrinogen stability being essentially equivalent to that of fibrin monomer or polymerized fibrin. SIGNIFICANCE: The low volume, high throughput assay has potential for use in understanding interactions with rare or mutant fibrin(ogen) variants.

Thrombin flux and wall shear rate regulate fibrin fiber deposition state during polymerization under flow.

Thrombin is released as a soluble enzyme from the surface of platelets and tissue-factor-bearing cells to trigger fibrin polymerization during thrombosis under flow conditions. Although isotropic fibrin polymerization under static conditions involves protofibril extension and lateral aggregation leading to a gel, factors regulating fiber growth are poorly quantified under hemodynamic flow due to the difficulty of setting thrombin fluxes. A membrane microfluidic device allowed combined control of both thrombin wall flux (10(-13) to 10(-11) nmol/mum(2) s) and the wall shear rate (10-100 s(-1)) of a flowing fibrinogen solution. At a thrombin flux of 10(-12) nmol/mum(2) s, both fibrin deposition and fiber thickness decreased as the wall shear rate increased from 10 to 100 s(-1). Direct measurement and transport-reaction simulations at 12 different thrombin flux-wall shear rate conditions demonstrated that two dimensionless numbers, the Peclet number (Pe) and the Damkohler number (Da), defined a state diagram to predict fibrin morphology. For Da < 10, we only observed thin films at all Pe. For 10 < Da < 900, we observed either mat fibers or gels, depending on the Pe. For Da > 900 and Pe < 100, we observed three-dimensional gels. These results indicate that increases in wall shear rate quench first lateral aggregation and then protofibril extension.

Advancing microarray assembly with acoustic dispensing technology.

In the assembly of microarrays and microarray-based chemical assays and enzymatic bioassays, most approaches use pins for contact spotting. Acoustic dispensing is a technology capable of nanoliter transfers by using acoustic energy to eject liquid sample from an open source well. Although typically used for well plate transfers, when applied to microarraying, it avoids the drawbacks of undesired physical contact with the sample; difficulty in assembling multicomponent reactions on a chip by readdressing, a rigid mode of printing that lacks patterning capabilities; and time-consuming wash steps. We demonstrated the utility of acoustic dispensing by delivering human cathepsin L in a drop-on-drop fashion into individual 50-nanoliter, prespotted reaction volumes to activate enzyme reactions at targeted positions on a microarray. We generated variable-sized spots ranging from 200 to 750 microm (and higher) and handled the transfer of fluorescent bead suspensions with increasing source well concentrations of 0.1 to 10 x 10(8) beads/mL in a linear fashion. There are no tips that can clog, and liquid dispensing CVs are generally below 5%. This platform expands the toolbox for generating analytical arrays and meets needs associated with spatially addressed assembly of multicomponent microarrays on the nanoliter scale.

Direct observation of membrane tethers formed during neutrophil attachment to platelets or P-selectin under physiological flow.

Adhesion and subsequent aggregation between neutrophils and platelets is dependent upon the initial binding of P-selectin on activated platelets to P-selectin glycoprotein ligand 1 (PSGL-1) on the microvilli of neutrophils. High speed, high resolution videomicroscopy of flowing neutrophils interacting with spread platelets demonstrated that thin membrane tethers were pulled from neutrophils in 32 +/- 4% of the interactions. After capture by spread platelets, neutrophil membrane tethers (length of 5.9 +/- 4.1 microm, n = 63) were pulled at an average rate of 6-40 microm/s as the wall shear rate was increased from 100-250 s(-1). The average tether lifetime decreased significantly (P < 0.001) from 630 to 133 ms as the shear rate was increased from 100 s(-1) (F(bond) = 86 pN) to 250 s(-1) (F(bond) = 172 pN), which is consistent with P-selectin/PSGL-1 bond dynamics under stress. Tether formation was blocked by antibodies against P-selectin or PSGL-1, but not by anti-CD18 antibodies. During neutrophil rolling on P-selectin at 150 s(-1), thin membrane tethers were also pulled from the neutrophils. The characteristic jerking motion of the neutrophil coexisted with tether growth (8.9 +/- 8.8 microm long), whereas tether breakage (average lifetime of 3.79 +/- 3.32 s) caused an acute jump in the rolling velocity, proving multiple bonding in the cell surface and the tether surface contact area. Extremely long membrane tethers (>40 microm) were sometimes pulled, which detached in a flow-dependent mechanism of microparticle formation. Membrane tethers were also formed when neutrophils were perfused over platelet monolayers. These results are the first visualization of the often hypothesized tethers that shield the P-selectin/PSGL-1 bond from force loading to regulate neutrophil rolling during inflammation and thrombosis.

Fluid flow stimulates tissue plasminogen activator secretion by cultured human endothelial cells.

Wall shear stress generated by blood flow may regulate the expression of fibrinolytic proteins by endothelial cells. Tissue plasminogen activator (tPA) and plasminogen activator inhibitor, type 1 (PAI-1) secretion by cultured human endothelial cells were not affected by exposure to venous shear stress (4 dynes/cm2). However, at arterial shear stresses of 15 and 25 dynes/cm2, the tPA secretion rate was 2.1 and 3.0 times greater, respectively, than the basal tPA secretion rate. PAI-1 secretion was unaffected by shear stress over the entire physiological range.

A series of cationic sterol lipids with gene transfer and bactericidal activity.

A family of cationic lipids was synthesized via direct amide coupling of spermine to the C-24 position of cholic acid analogs. Four monosubstituted spermines and a bis-substituted spermine were evaluated as plasmid transfection reagents, as bacteriostatic agents, and as bactericidal agents. The incorporation of a double bond in the sterol moiety enhanced transfection efficiency significantly and produced two compounds with little cytotoxicity and transfection potency comparable to Lipofectamine2000. Inclusion of the double bond had no effect on the general trend of increasing bactericidal activity with increasing sterol hydrophobicity. Co-formulation of the most hydrophilic of the compounds with its bis-substituted analogue led to enhancement in transfection activity. The bis-substituted compound, when tested alone, emerged as the most bacteriostatic compound in the family with minimum inhibitory concentrations (MIC) of 4 microM against Bacillus subtilis and 16 microM against Escherichia coli and therapeutic indexes (minimum hemolytic concentration/minimum inhibitory concentration) of 61 and 15, respectively. Cationic lipids can be optimized for both gene delivery and antibacterial applications by similar modifications.

Enzyme microarrays assembled by acoustic dispensing technology.

Miniaturizing bioassays to the nanoliter scale for high-throughput screening reduces the consumption of reagents that are expensive or difficult to handle. Through the use of acoustic dispensing technology, nanodroplets containing 10 microM ATP (3 microCi/microL (32)P) and reaction buffer in 10% glycerol were positionally dispensed to the surface of glass slides to form 40-nL compartments (100 droplets/slide) for Pim1 (proviral integration site 1) kinase reactions. The reactions were activated by dispensing 4 nL of various levels of a pyridocarbazolo-cyclopentadienyl ruthenium complex Pim1 inhibitor, followed by dispensing 4 nL of a Pim1 kinase and peptide substrate solution to achieve final concentrations of 150 nM enzyme and 10 microM substrate. The microarray was incubated at 30 degrees C (97% R(h)) for 1.5 h. The spots were then blotted to phosphocellulose membranes to capture phosphorylated substrate. With phosphor imaging to quantify the washed membranes, the assay showed that, for doses of inhibitor from 0.75 to 3 microM, Pim1 was increasingly inhibited. Signal-to-background ratios were as high as 165, and average coefficients of variation for the assay were approximately 20%. Coefficients of variation for dispensing typical working buffers were under 5%. Thus, microarrays assembled by acoustic dispensing are promising as cost-effective tools that can be used in protein assay development.

Hemodynamic force triggers rapid NETosis within sterile thrombotic occlusions.

Essentials Neutrophil extracellular traps (NETs) are generated during thrombosis and sepsis. The effect of hemodynamics on NETosis during sterile thrombosis was studied using microfluidics. Pressure gradients > 70 mmHg per mm-clot across sterile occlusions drive shear-induced NETosis. High interstitial hemodynamic forces trigger rapid NET release. SUMMARY: Background Neutrophil extracellular traps (NETs) are released when neutrophils encounter infectious pathogens, especially during sepsis. Additionally, NETosis occurs during venous and arterial thrombosis, disseminated intravascular coagulation, and trauma. Objective To determine whether hemodynamic forces trigger NETosis during sterile thrombosis. Methods NETs were imaged with Sytox Green during microfluidic perfusion of activated factor XII-inhibited or thrombin-inhibited human whole blood over fibrillar collagen (with or without tissue factor). Results For perfusions at initial inlet venous or arterial wall shear rates (100 s-1 or 1000 s-1 ), platelets rapidly accumulated and occluded microchannels with subsequent neutrophil infiltration under either flow condition; however, NETosis was detected only in the arterial condition. The level of shear-induced NETs (SINs) at 30 min was > 150-fold higher in the arterial condition in the absence of thrombin and > 80-fold greater in the presence of thrombin than the level in the venous condition. With or without thrombin, venous perfusion for 15 min generated no NETs, but an abrupt shift-up to arterial perfusion triggered NETosis within 2 min, NETs eventually reaching levels 15 min later that were 60-fold greater than that in microchannels without perfusion shift-up. SINs contained citrullinated histone H3 and myeloperoxidase, and were DNase-sensitive, but were not blocked by inhibitors of platelet-neutrophil adhesion, high-mobility group protein box 1-receptor for advanced glycation end products interaction, cyclooxygenase, ATP/ADP, or peptidylarginine deiminase 4. For measured pressure gradients exceeding 70 mmHg per millimeter of clot across NET-generating occlusions to drive interstitial flow, the calculated fluid shear stress on neutrophils exceeded the known lytic value of 150 dyne cm-2 . Conclusions High interstitial hemodynamic forces can drive physically entrapped neutrophils to rapidly release NETs during sterile occlusive thrombosis.

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.

Tissue plasminogen activator (tPA) inhibits plasmin degradation of fibrin. A mechanism that slows tPA-mediated fibrinolysis but does not require alpha 2-antiplasmin or leakage of intrinsic plasminogen.

Thrombolysis is dramatically slower when high concentrations of lytic agent are used. This paradoxical observation, first described as "plasminogen steal," was originally believed to be due to depletion of extrinsic plasminogen and consequent leaching of clot-bound plasminogen. We report that administration of increasing concentrations of recombinant human tissue plasminogen activator (tPA) to fibrin gels resulted in lysis rates that displayed a maximum, with significantly slower rates found at higher tPA, regardless of whether plasminogen was supplied extrinsically or intrinsically. A similar maximum in lysis rates was observed in a system lacking an extrinsic phase when plasminogen was added to fibrin suspensions preincubated with increasing tPA. Thus, intrinsic plasminogen leakage and alpha 2-antiplasmin were not required for the decreased lysis at high tPA. No maximum was observed for increasing concentrations of urokinase. Using fibrin suspensions or gels preincubated with tPA before addition of plasmin, we report that tPA, but not urokinase, caused a dose-dependent inhibition of the fibronolytic action of plasmin. With respect to optimal dosage schemes and the design of novel lytic agents, these findings indicate that (a) there exists a biochemical mechanism against minimizing reperfusion time with increasing tPA dosages and (b) the fibrin affinity of tPA may cause reduced fibrinolysis by plasmin.

Nuclear targeting peptide scaffolds for lipofection of nondividing mammalian cells.

Lipofection of nondividing cells is inefficient because much of the transfected DNA is retained in endosomes, and that which escapes to the cytoplasm enters the nucleus at low rates. To improve the final rate-limiting step of nuclear import, we conjugated a nonclassical nuclear localization signal (NLS) containing the M9 sequence of heterogeneous nuclear ribonucleoprotein (hnRNP) A1, to a cationic peptide scaffold derived from a scrambled sequence of the SV40 T-antigen consensus NLS (ScT). The ScT was added to improve DNA binding of the M9 sequence. Lipofection of confluent endothelium with plasmid complexed with the M9-ScT conjugate resulted in 83% transfection and a 63-fold increase in marker gene expression. The M9-ScT conjugate localized fluorescent plasmid into the nucleus of permeabilized cells, and addition of the nuclear pore blocker wheat germ agglutinin prevented nuclear import. This method of gene transfer may lead to viral- and lipid-free transfection of nondividing cells.

Soluble fibrin causes an acquired platelet glycoprotein VI signaling defect: implications for coagulopathy.

Essentials Collagen and thrombin when used simultaneously generate highly activated platelets. The effect of thrombin stimulation on subsequent glycoprotein VI (GPVI) function was observed. Soluble fibrin, but not protease activated receptor (PAR) activation, prevented GPVI activation. Circulating soluble fibrin in coagulopathic blood may cause an acquired GPVI signaling defect. SUMMARY: Background In coagulopathic blood, circulating thrombin may drive platelet dysfunction. Methods/Results Using calcium dye-loaded platelets, the effect of thrombin exposure and soluble fibrin generation on subsequent platelet GPVI function was investigated. Exposure of apixaban-treated platelet-rich plasma (12% PRP) to thrombin (1-10 nm), but not ADP or thromboxane mimetic U46619 exposure, dramatically blocked subsequent GPVI activation by convulxin, collagen-related peptide or fibrillar collagen. Consistent with soluble fibrin multimerizing and binding GPVI, the onset of convulxin insensitivity required 200-500 s of thrombin exposure, was not mimicked by exposure to PAR-1/4 activating peptides, was not observed with washed platelets, and was blocked by fibrin polymerization inhibitor (GPRP) or factor XIIIa inhibitor (T101). PAR-1 signaling through Gαq was not required because vorapaxar blocked thrombin-induced calcium mobilization but had no effect on the ability of thrombin to impair GPVI-signaling. Convulxin insensitivity was unaffected by the metalloprotease inhibitor GM6001 or the αIIb ß3 antagonist GR144053, indicating negligible roles for GPVI shedding or αIIb ß3 binding of fibrin. Thrombin treatment of washed platelets resuspended in purified fibrinogen also produced convulxin insensitivity that was prevented by GPRP. Exposure of apixaban/PPACK-treated whole blood to thrombin-treated fibrinogen resulted in > 50% decrease in platelet deposition in a collagen microfluidic assay that required soluble fibrin assembly. Conclusions Conversion of only 1% plasma fibrinogen in coagulopathic blood would generate 90 nm soluble fibrin, far exceeding ~1 nmGPVI in blood. Soluble fibrin, rather than thrombin-induced platelet activation throuh PAR-1 and PAR-4, downregulated GPVI-signaling in response to stimuli, and may lead to subsequent hypofunction of endogenous or transfused platelets.

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.

Transport physics and biorheology in the setting of hemostasis and thrombosis.

The biophysics of blood flow can dictate the function of molecules and cells in the vasculature with consequent effects on hemostasis, thrombosis, embolism, and fibrinolysis. Flow and transport dynamics are distinct for (i) hemostasis vs. thrombosis and (ii) venous vs. arterial episodes. Intraclot transport changes dramatically the moment hemostasis is achieved or the moment a thrombus becomes fully occlusive. With platelet concentrations that are 50- to 200-fold greater than platelet-rich plasma, clots formed under flow have a different composition and structure compared with blood clotted statically in a tube. The platelet-rich, core/shell architecture is a prominent feature of self-limiting hemostatic clots formed under flow. Importantly, a critical threshold concentration of surface tissue factor is required for fibrin generation under flow. Once initiated by wall-derived tissue factor, thrombin generation and its spatial propagation within a clot can be modulated by γ'-fibrinogen incorporated into fibrin, engageability of activated factor (FIXa)/activated FVIIIa tenase within the clot, platelet-derived polyphosphate, transclot permeation, and reduction of porosity via platelet retraction. Fibrin imparts tremendous strength to a thrombus to resist embolism up to wall shear stresses of 2400 dyne cm(-2) . Extreme flows, as found in severe vessel stenosis or in mechanical assist devices, can cause von Willebrand factor self-association into massive fibers along with shear-induced platelet activation. Pathological von Willebrand factor fibers are A Disintegrin And Metalloprotease with ThromboSpondin-1 domain 13 resistant but are a substrate for fibrin generation due to FXIIa capture. Recently, microfluidic technologies have enhanced the ability to interrogate blood in the context of stenotic flows, acquired von Willebrand disease, hemophilia, traumatic bleeding, and drug action.

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

UNLABELLED: 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. SUMMARY: Background Protein disulfide isomerases (PDIs) may regulate thrombus formation in vivo, although the sources and targets of PDIs are not fully understood. Methods and results Using click chemistry to link anti-CD61 and a C-terminal azido disulfide-linked peptide construct with a quenched reporter, we developed a fluorogenic platelet-targeting antibody (PDI-sAb) for thiol reductase activity detection in whole blood under flow conditions. PDI-sAb was highly responsive to various exogenous reducing agents (dithiothreitol, glutathione and recombinant PDI) and detected thiol reductase activity on P-selectin/phosphatidylserine-positive platelets activated with convulxin/PAR1 agonist peptide, a signal partially blocked by PDI inhibitors and antibody. In a microfluidic thrombosis model using 4 µg mL(-1) corn trypsin inhibitor-treated human blood perfused over collagen (wall shear rate = 100 s(-1) ), the PDI-sAb signal increased mostly over the first 200 s, whereas platelets continually accumulated for over 500 s, indicating that primary adhesion to collagen, but not secondary aggregation, was correlated with the PDI-sAb signal. Rutin and the PDI blocking antibody RL90 reduced platelet adhesion and the PDI-sAb signal only when thrombin production was inhibited with PPACK, suggesting limited effects of platelet thiol isomerase activity on platelet aggregation on collagen in the presence of thrombin. With anti-mouse CD41 PDI-sAb used in an arteriolar laser injury model, thiol reductase activity was localized in the core of growing thrombi where platelets displayed P-selectin and were in close proximity to disrupted endothelium. Conclusion PDI-sAb is a sensitive and real-time reporter of platelet- and vascular-derived disulfide reduction that targets clots as they form under flow to reveal spatial gradients.

Inhibition of nitric oxide but not prostacyclin prevents poststenotic dilatation in rabbit femoral artery.

BACKGROUND: Poststenotic dilatation (PSD) occurs in a low-pressure region where recirculation eddies oscillate in size during the cardiac cycle. NO may be an important mediator of PSD. METHODS AND RESULTS: Femoral arteries of 7 adult male New Zealand White rabbits were stenosed bilaterally to achieve a diameter reduction of 70. 9+/-6.7% (n=14). At the time of stenosis, the adventitia of one of the arteries was coated with 1 mmol/L of NG-nitro-L-arginine methyl ester (L-NAME) in 22% (wt/vol) Pluronic gel, while the contralateral vessel was coated with gel without L-NAME. In stenosed femoral arteries that were treated with gel without L-NAME, a maximum PSD of 30.99+/-7.92% (n=7) was observed in polymer casts at 3 days relative to the mean proximal diameter of 1.57+/-0.25 mm at a position 12 mm upstream of each stenosis. In contrast, the vessels treated with L-NAME exhibited a maximum PSD of only 7.16+/-8.81% (n=7) relative to the mean proximal diameter of 1.55+/-0.16 mm. L-NAME caused a 76. 9% reduction (P<0.001, n=7) of PSD. Similarly, NG-monomethyl-L-arginine 1 mmol/L and NG-nitro-L-arginine 10 micromol/L attenuated PSD by 57.5% (P<0.001, n=6) and 63.9% (P<0.05, n=6), respectively. Indomethacin 10 micromol/L caused no reduction in PSD. Arterial rings obtained from the poststenotic region were more sensitive and responsive to acetylcholine than those obtained proximal to the stenosis. CONCLUSIONS: NO, but not prostacyclin, is a major mediator of PSD.