(H-298) Platelets and von Willebrand factor after exposure to turbulent but low shear flow with implications in mechanical circulatory support

Introduction::
Mechanical circulatory support (MCS) is an important treatment option for patients with heart failure. Despite its many benefits, bleeding complications remain an unsolved problem. Almost all patients with MCS implantations develop the bleeding disorder, acquired von Willebrand syndrome (aVWS). aVWS is characterized by the loss of high molecular weight von Willebrand factor (VWF) multimers.




VWF is a large multimeric protein that plays an important role in primary hemostasis. Due to cleavage of VWF multimers, there is a large range of sizes in circulation, with increasing function in hemostasis with increasing size. Platelets also play a critical role in hemostasis. Upon vascular injury, platelets adhere to the sub-endothelial matrix via binding of VWF. This adhesion leads to intracellular signaling and activation of the platelet. Once activated, platelets undergo conformational shape changes as well as degranulation, releasing P-selectin.




Though the current dogma suggests that high shear stress results in the cleavage of VWF, activation of platelets, and thus bleeding, high shear alone may not be sufficient to explain the degree of VWF loss seen in patients. This drives our group to evaluate other potential contributors such as the role of turbulence.




Transverse aortic constriction (TAC) is a surgical model in mice that places a permanent constriction around the transverse aorta. The consistency of the constriction is maintained by tying a suture around various needle diameters alongside the aorta and then removing the needle. Here, we compare laminar and turbulent flow found mild and severe stenoses to demonstrate bleeding after exposure to turbulence.




Materials and Methods::
C57BL/6 mice obtained from Jackson Laboratory were used for experimentation. TAC surgeries were performed with varying degrees of constriction. A larger 24g needle for a mild stenosis and a smaller 26g needle for a severe stenosis. A control procedure was performed with a 0% stenosis. Post-surgery, blood was drawn retro-orbitally into heparinized capillary tubes at 24 hours and 7 days and processed to assess VWF cleavage and platelet activation.




SDS-agarose gel electrophoresis was performed using a 1.2% agarose gel. Electrophoresis was conducted for 16 hours and then transferred to polyvinylidene difluoride membranes for 1 hour. Membranes were blocked for 1 hour and incubated in HRP-conjugated anti-human VWF antibody for 1 hour. Following antibody incubation, membranes were developed with a gel doc machine.




Antibodies (1:100) were added to each sample. As a negative control, control sham mice platelet rich plasma (PRP) was used. For a positive control, 10 mM of thrombin receptor activating peptide (TRAP), a known agonist, was used. Following incubation in antibodies for 30 min, platelets were fixed using 1.5% paraformaldehyde and read using a flow cytometer machine.




Human peripheral whole blood was drawn and centrifuged to first separate the red blood cells from platelet-rich plasma (PRP). PRP was centrifuged again to obtain platelet-poor plasma (PPP). PPP was exposed to varying rotational speeds (34, 112, 150 rad/s) in the vortexer. Multimer cleavage analysis was performed as described above. Along with cleavage assays, platelet activation was also evaluated using flow cytometry as described above.




Results, Conclusions, and Discussions:: The TAC surgery mouse model was used to demonstrate bleeding complications after exposure to turbulence. Post-surgery, the degree of VWF cleavage and platelet activation were measured at two different time points after recovery. At the 24 hours time point, VWF cleavage and platelet activation were only statistically significant when the control sham mice were compared to mice with severe stenoses. The mice with mild stenoses did not exhibit significant differences to the control sham mice in terms of VWF cleavage and platelet activation. (Figure 1). The two markers used in flow cytometry detect the activated form of integrin a_IIbb₃ via Jon/A and release of P-selectin via CD62. Platelet activation leads to conformational changes to the a_IIbb₃ complex on platelet surfaces. P-selectin is expressed on a-granule membranes inside resting platelets but translocates to the platelet surface after activation.

As a systematic way to investigate the observations we saw in our mouse model, the vortexer assay was used. This vortexer model allows turbulence to be studied with low sample volumes in the absence of high shear stress. Flow was quantified through CFD and validated with high-speed images of physical experiments to quantify the shear stress in the system. Differing rotational speeds on the vortexer simulate different flow environments. A rotational speed of 34 rad/s is laminar, 112 rad/s is transitional, and 150 rad/s is turbulent. After exposure to the various rotational speeds on the vortexer, no loss of high molecular weight multimers (HMWM) was observed in conditions of laminar and transitional shear. However, in turbulent conditions, there was a loss of HMWM, with an increasing reduction in multimer size as exposure time increased. (Figure 2).

Platelets, in addition to VWF, also affect the hemostatic response after exposure to turbulent flow. PRP was vortexed at the same rotational speeds as described above and evaluated for integrin a_IIbb₃ activation via PAC-1 and P-selectin release from granules. As displayed in histogram overlays (Figure 3), there was an increase in activation with increased rotational speed.

Our findings address a key part in the mechanisms underlying the bleeding phenotype of MCS patients.



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