High-shear stress sensitizes platelets to subsequent low-shear conditions

Abstract

Individuals with mechanical heart valve implants are plagued by flow-induced thromboembolic complications, which are undoubtedly caused by platelet activation. Flow fields in or around the affected regions involve brief exposure to pathologically high-shear stresses on the order of 100 to 1000 dyne/cm(2). Although high shear is known to activate platelets directly, their subsequent behavior is not known. We hypothesize that the post-high-shear activation behavior of platelets is particularly relevant in understanding the increased thrombotic risk associated with blood-recirculating prosthetic cardiovascular devices. Purified platelets were exposed to brief (5-40 s) periods of high-shear stress, and then exposed to longer periods (15-60 min) of low shear. Their activation state was measured using a prothrombinase-based assay. Platelets briefly exposed to an initial high-shear stress (e.g., 60 dyne/cm(2) for 40 s) activate a little, but this study shows that they are now sensitized, and when exposed to subsequent low shear stress, they activate at least 20-fold faster than platelets not initially exposed to high shear. The results show that platelets in vitro exposed beyond a threshold of high-shear stress are primed for subsequent activation under normal cardiovascular circulation conditions, and they do not recover from the initial high-shear insult.

FIGURE 1

Pre-exposure to high-shear stress, 40 s duration. Platelets were pre-exposed to shear stresses of 1 (control), and 70 dyne/cm², as shown in the top bar, followed by exposure to 1 dyne/cm² for 59 min. The means of four experiments are shown ±SEM.

FIGURE 2

Pre-exposure to varying shear stress, 40 s duration. Platelets were pre-exposed to shear stresses of 1 (control), 50, 60, and 70 dyne/cm², as shown in the top bar, followed by exposure to 1 dyne/cm² for 14 min. The means of nine experiments are shown ±SEM.

FIGURE 3

Pre-exposure to fixed high-shear stress for varying duration. Platelets were pre-exposed to 70 dyne/cm² shear stress for 20, 30, and 40 s, as shown in the top bar, followed by 1 dyne/cm² for 14 min. Means of 11 experiments are shown ±SEM.

FIGURE 4

Sensitization threshold. Platelet activation rates after high-shear pre-exposure (Figs. 1 and 2, and Table 1) are plotted against the time integral of the initial high-shear stress.

FIGURE 5

Comparison of PAS and platelet lysis. Platelets were pre-exposed to 70 dyne/cm² shear stress for 40 s, followed by 1 dyne/cm² for 14 min. Samples were assessed for PAS, and for platelet lysis by LDH release.

FIGURE 6

Intracellular Ca²⁺ depletion and platelet sensitization. Platelets treated with 20 µM BAPTA and untreated platelets were pre-exposed to 70 dyne/cm² shear stress for 40 s, followed by 1 dyne/cm² for 14 min.

FIGURE 7

PMPs and sensitization. Comparison of total (platelet + microparticle, black) and microparticle (white) activity after single 40-s exposure to 70 dyne/cm² and subsequent 15-min exposure to 1 dyne/cm².