Activity

Both full-length and cleaved forms of YRS were taken up by platelets in vitro and stored in the α-granules. The N-terminal YRS fragment generated by proteolytic cleavage had monocyte activation comparable to that of the constitutive-active mutant YRS (YRS

) previously reported.

Platelets take up both full-length YRS and the active form of cleaved YRS fragment from the plasma. The cleaved, N-terminal YRS fragment stored in α-granules may have potential to activate monocytes.

Platelets take up both full-length YRS and the active form of cleaved YRS fragment from the plasma. The cleaved, N-terminal YRS fragment stored in α-granules may have potential to activate monocytes.

Hemostatic clots have a P-selectin positive platelet core covered with a shell of P-selectin negative platelets.

To develop a new human blood microfluidic assay to interrogate core/shell mechanics.

A 2-stage assay perfused whole blood over collagen/± tissue factor (TF) for 180seconds at 100s

wall shear rate, followed by buffer perfusion at either 100s

(venous) or 1000s

(arterial). This microfluidic assay used an extended channel height (120µm), allowing buffer perfusion well before occlusion.

Clot growth on collagen stopped immediately with buffer exchange, revealing ~10% reduction in platelet fluorescence intensity (at 100s

) and ~30% (at 1000s

) by 1200seconds. Thrombin generation (on collagen/TF) reduced erosion at either buffer flow rate. P-selectin-positive platelets were stable (no erosion) against 1000s

, in contrast to P-selectin negative platelets. Thrombin inhibition (with D-Phe-Pro-Arg-CMK) reduced the number of P-selectin-positive platelets and lowered thrombus stability through the reduction of P-selectin-positive platelets. Interestingly, fibrin inhibition (with H-Gly-Pro-Arg-Pro-OH acetate salt) increased the number of P-selectin-positive platelets but did not lower stability, suggesting that fibrin was only in the core region. Thromboxane inhibition reduced P-selectin-positive platelets and caused a nearly 60% reduction of the clot at arterial buffer flow. P2Y1 antagonism reduced clot size and the number of P-selectin-positive platelets and reduced the stability of P-selectin-negative platelets.

The 2-stage assay (extended channel height plus buffer exchange) interrogated platelet stability using human blood. Under all conditions, P-selectin-positive platelets never left the clot.

The 2-stage assay (extended channel height plus buffer exchange) interrogated platelet stability using human blood. Under all conditions, P-selectin-positive platelets never left the clot.

Blood clotting in humans is initiated by the binding of tissue factor to activated coagulation factor VII (FVIIa) in the plasma. Previous studies have reported that hepsin and factor VII (FVII)-activating protease are responsible for generating FVIIa.

We aimed to identify other proteases that may activate FVII using zebrafish as a model.

We screened 179 genes encoding serine protease domains using the piggyback knockdown method to identify genes involved in the activation of zebrafish Fvii. A prolonged kinetic prothrombin time (kPT) assay was used to detect gene knockdown effects.

In the primary screen, 21 genes showed prolonged kPT. In the secondary screen, 14 of 21 genes showed positive results. In the tertiary screen, all 14 genes showed prolonged kPT. These 14 genes were knocked down again to estimate relative levels of zebrafish Fviia. find more Six genes, including known genes, such as

and novel

and

(

), showed lower Fviia levels. Fvii levels were affected only by the knockdown of

and not by the knockdown of the other five genes.

and

are involved in generating Fviia. We hypothesize that prostasin exerts serine protease activity directly or indirectly to activate Fvii. As Hgfb has a mutated serine protease domain, it may not cleave Fvii but may bind to Fvii to induce autoactivation. The approach developed here may be extended to design other large-scale knockdown screens.

Prostasin and hgfb are involved in generating Fviia. We hypothesize that prostasin exerts serine protease activity directly or indirectly to activate Fvii. As Hgfb has a mutated serine protease domain, it may not cleave Fvii but may bind to Fvii to induce autoactivation. The approach developed here may be extended to design other large-scale knockdown screens.

The efficacy and safety of thrombomodulin alfa (TM-α), a cofactor protein promoting thrombin-mediated protein C activation, have been examined in a phase 3 randomized, double-blinded, parallel-group trial in Japan. We have previously reported that TM-α is noninferior to heparin for the resolution of disseminated intravascular coagulation (DIC).

To investigate the basis for the efficacy of TM-α in the phase 3 clinical trial in Japan through post hoc analysis of coagulation and fibrinolysis parameters.

The 227 patients of the full analysis set population described in the original phase 3 trial in Japan were included in this analysis. Changes in parameters between before and after TM-α or heparin administration in each of the two patient groups, with underlying diseases of either hematologic malignancy or infection, were studied separately and results were compared between TM-α and heparin treatment groups in a post hoc manner.

TM-α administration did not prolong activated partial thromboplastin time but significantly decreased thrombin-antithrombin complex levels compared with heparin treatment. TM-α administration reduced consumption of endogenous anticoagulants such as antithrombin and protein C by DIC, compared with the heparin group. DIC scores were decreased in both TM-α and heparin groups during the 6-day treatment.

TM-α can alleviate intravascular coagulation and consumption of anticoagulants without extending coagulation times. This may be associated with the relatively low risk of bleeding during TM-α treatment.

TM-α can alleviate intravascular coagulation and consumption of anticoagulants without extending coagulation times. This may be associated with the relatively low risk of bleeding during TM-α treatment.