The PLATELET INTEGRIN αIIbβ3 CHANGES FROM A LOWER- to A Higher-AFFINITY STATE DURING INTERACTION with FIBRINOGEN
Authors: Litvinov Rustem I. , Barsegov Valeri , Mekler Andrey , Weisel John W. , Bennett Joel S. 
Published: October 2019

Abstract
Abstract 1130
The integrin αIIbβ3 (GPIIb-IIIa) plays an essential role in platelet adhesion and cohesion by binding to a number of adhesive ligands including fibrinogen, von Willebrand factor, fibronectin, and vitronectin. Following ligand binding, αIIbβ3-mediated intracellular signaling is thought to stabilize platelet adhesive interactions, cause platelet spreading, and initiate clot retraction. Here we investigated the time course of interactions between purified αIIbβ3 and its major ligand fibrinogen when both were firmly attached to apposed surfaces, mimicking αIIbβ3-mediated platelet adhesive interactions. To measure the mechanical resistance of individual αIIbβ3-fibrinogen complexes under a constant tensile force, we used a previously described optical trap force clamp system (Litvinov et al., Biophys. J., 2011, 100, 165). Briefly, we trapped a fibrinogen-coated microscopic bead in a focused laser beam and intermittently brought it into contact with an αIIbβ3-coated silica pedestal. Repeated touching and separation of the bead and pedestal occurred with a compressive force of 20 pN and a constant pulling force of 50 pN. When the duration of contact between interacting molecules on the bead and pedestal was varied from 0.1s to 2s, the probability of αIIbβ3-fibrinogen interaction was time-dependent. This enabled us to extract two-dimensional kinetic parameters, i.e., how fast the αIIbβ3-fibrinogen complex forms and dissociates at the interface without the application of an external tensile force (zero-force kinetics). To convert the kinetic parameters to absolute values, we determined the density of αIIbβ3 molecules on the pedestal surface capable of binding 125I-fibrinogen to be ≈3,000 molecules per μm2. We found that the reactive αIIbβ3 exists in at least two states that differ significantly in their on-rates (kon1 =8×10−5 and kon2 =5.6×10−4 μm2/s), off-rates (koff1 =1.56 and koff2 =1.70 1/s) and affinities (Kd1 =2×104 and Kd2 =3×103 1/μm2) for fibrinogen. In the absence of αIIbβ3 activators, the interacting molecules reached a stable equilibrium in which 85% of the complexes were in the lower affinity form. In the presence of the activator Mn2+, the proportion of lower affinity forms decreased to 60%. Mn2+ also changed the on-rates (kon1 =3.38×10−5 and kon2 =9.9×10−4 μm2/s), off-rates (koff1 =2.99 and koff2 =4.82 1/s) and affinities (Kd1 =9×104 and Kd2 =5×103 1/μm2). We then tested whether the ratio between the two integrin activation states and the mechanical stability of the αIIbβ3-fibrinogen complex depends on the duration of contact between the interacting surfaces. As the contact duration was prolonged from 0.1s to 2s, the αIIbβ3-fibrinogen bond lifetime increased from shorter lifetimes (<2s) corresponding to the low-affinity state to longer lifetimes (up to 50s) corresponding to the high-affinity state. Moreover, the rate of transition from low- to high-affinity increased ∼3-fold, whereas the reverse rate decreased 1.5–2-fold, resulting in an overall 6-fold increase in the equilibrium rate constant. This means that reversible conformational transitions of αIIbβ3 from low- to high affinity states occur during its interaction with fibrinogen with the equilibrium gradually shifting toward the higher affinity state as contact duration increases. Thus, the strength of αIIbβ3-fibrinogen interactions is time-dependent due to a progressive increase in the affinity of the αIIbβ3-fibrinogen complex during the course of the interaction. These results provide kinetic and thermodynamic characteristics of the adhesive interaction between αIIbβ3 and fibrinogen at the single-molecule level and provide evidence for a direct fibrinogen-induced change in αIIbβ3 conformation as a potential mechanism for the initiation of αIIbβ3-mediated outside-in signaling in platelets.

Disclosures:
No relevant conflicts of interest to declare.