Force Spectroscopy of Interactions of the Integrin AlphaIIbbeta3 with Fibrin and Fibrinogen

Abstract

We compared the probability, strength, and specificity of the interactions of integrin alphaIIbbeta3 with fibrin versus fibrinogen. Briefly, a bead coated with purified alphaIIbbeta3 was trapped by a laser beam and repeatedly brought into contact with surface-attached fibrinogen, monomeric fibrin, or a naturally-formed hydrated fibrin fiber. When alphaIIbbeta3-ligand complexes were detected, the rupture forces were measured. Force histograms for alphaIIbbeta3-fibrinogen interactions could be segregated into moderate (20-60 pN) and strong (>60 pN) interactions. Monomeric fibrin displayed a higher cumulative probability of interacting with alphaIIbbeta3 and a greater binding strength. alphaIIbbeta3-fibrin interactions were also less sensitive to inhibition by alphaIIbbeta3 antagonists, suggesting that they had different binding specificity. Both fibrinogen and fibrin interactions with alphaIIbbeta3 were partially inhibited by RGD-containing peptides, suggesting the existence of common RGD-containing binding motifs. This assumption was supported using the fibrin variants alphaD97E or alphaD574E whose mutated RGD motifs were less reactive with alphaIIbbeta3 than those of wild type fibrin. Monomeric fibrin made from a fibrinogen lacking the gammaC alphaIIbbeta3-binding motif was more reactive with alphaIIbbeta3 than the parent fibrinogen, suggesting that this binding motif is less important in fibrin. Polymeric fibrin displayed a rupture force profile similar to fibrinogen and monomeric fibrin with moderate (20-60 pN) and strong (>60 pN) forces that peaked at 70-80 pN. alphaIIbbeta3 antagonists, cycloRGD-peptide, and the gammaC-dodecapeptide each produced a sharp drop in the medium-to-high force range. These results demonstrate that surface-bound fibrinogen and monomeric, as well as polymeric, fibrin are highly reactive with the platelet receptor alphaIIbbeta3. Fibrin is more reactive than fibrinogen in terms of its binding probability and it has greater binding strength. Fibrin binding to alphaIIbbeta3 was also less sensitive to alphaIIbbeta3 inhibitors, suggesting that fibrin and fibrinogen have distinct binding specificity.