Topographic Cues Reveal Two Distinct Spreading Mechanisms in Blood Platelets

Abstract

Blood platelets are instrumental in blood clotting and are thus heavily involved in early wound closure. After adhering to a substrate they spread by forming protrusions like lamellipodia and filopodia. However, the interaction of these protrusions with the physical environment of platelets while spreading is not fully understood. Here we dynamically image platelets during this spreading process and compare their behavior on smooth and on structured substrates. In particular we analyze the temporal evolution of the spread area, the cell morphology and the dynamics of individual filopodia. Interestingly, the topographic cues enable us to distinguish two spreading mechanisms, one that is based on numerous persistent filopodia and one that rather involves lamellipodia. Filopodia-driven spreading coincides with a strong response of platelet morphology to the substrate topography during spreading, whereas lamellipodia-driven spreading does not. Thus, we quantify different degrees of filopodia formation in platelets and the influence of filopodia in spreading on structured substrates.

Dynamics of force generation by spreading platelets

“…A full description of the isolation procedure can be found elsewhere. 11,30,31 Briefly, 4 mL of platelet concentrate was mixed with prostaglandin E 1 (Cayman Chemical Company, Ann Harbor, MI, USA; final concentration 2.6 mg mL À1 ) and centrifuged at 480 Â g and 21 1C for 20 min. The supernatant was removed and the cells resuspended in PSG (Pipes Saline Glucose: 5 mM PIPES, 145 mM NaCl, 4 mM KCl, 1 mM MgCl 2 Á6H 2 O, 5 mM glucose, and 0.05 mM Na 2 HPO 4 ).…”

Human blood platelets contract in perpendicular direction to shear flow

“…The isolation procedure has been previously described elsewhere. 5,16,27 Briefly, 4 mL of blood plasma was mixed with prostaglandin E 1 (Cayman Chemical Company, Ann Harbor, MI, USA; final concentration 2.6 mg mL À1 ). The solution was centrifuged for 20 min at 480Âg and 21 1C and the supernatant was removed.…”

“…This resulted in a dilution of the inlet thrombin concentration from 40 units per mL to a final outlet concentration of 4 units per mL, which is well within the physiological range, 30 thus ensuring complete activation of the platelets. 6,27 This solution was fed into the measuring chamber. The syringes were driven by a syringe pump system (neMESYS, Centoni GmbH, Korbuß en, Germany).…”

Morphometric analysis of spread platelets identifies integrin αIIbβ3-specific contractile phenotype

“…The morphology of spreading platelets has been extensively studied in vitro. Platelet spreading on glass proceeds fast (1-2 min) and independent of adhesion protein identity [4][5][6] .…”