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Interdisciplinary Seminar on Mathematical and Computational Modeling

Rustem I. Litvinov, PhD Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, PA

Tuesday, November 14, 2017
  2:10–3:30 p.m.


Location: Surge Building 284
  Parking Information

Category: Seminar

Description:

Although the integrin aIIbb3 mediates platelet spreading on surfaces artificially-coated with fibrinogen, the physiologic relevance of this phenomenon is not clear.  By contrast, the interaction of aIIbb3 with fibrin is responsible for clot retraction, an event important for efficient hemostasis in the hemodynamic environment of flowing blood. Moreover, whereas resting aIIbb3 can interact with immobilized fibrinogen, platelet-fibrin interactions are mediated by activated aIIbb3. Lastly, the efficacy of aIIbb3 antagonists with regard to clot contraction versus platelet aggregation mediated by aIIbb3 binding to fibrinogen interactions is different, perhaps because additional aIIbb3 binding sites are exposed when fibrinogen is converted to fibrin. Here, we have compared nanomechanical measurements of the direct interaction of aIIbb3 with fibrin and fibrinogen in order to explain these differential effects. We used optical laser tweezers-based force spectroscopy for these measurements.  Briefly, a bead covalently coated with purified aIIbb3 was captured by a focused laser beamand repeatedly brought into contact with surface-attached fibrinogen, monomeric fibrin, or a naturally-formed hydrated fibrin fiber at the edge of a plasma clot. When an aIIbb3-ligand complex was detected, the nanomechanical force required to dissociate the complex was measured at piconewton resolution. By analyzing distributions of these rupture forces, we were able to determine the overall reactivity and the strength of the interactions between the interacting protein pairs. Beside native fibrinogen, experiments were performed with recombinant fibrinogen variants as well. Monomeric fibrin displayed higher cumulative probability of interacting with aIIbb3 (a greater force-free on-rate) and binding strength (a smaller forced off-rate). aIIbb3-fibrin interactions were less sensitive to the effects of abciximab and eptifibatide compared to fibrinogen, suggesting that they had different binding specificity. Both fibrinogen- and fibrin-integrin interactions were partially blocked by the RGD-containing peptides, suggesting the existence of common RGD integrin-binding sites. This assumption was supported using fibrin variants αD97E or αD574E with impaired RGD motifs, which were less reactive with aIIbb3 than the wild type fibrin. Monomeric fibrin made from a homodimeric fibrinogen splice variant of the g chain (g´) that lacks the γC aIIbb3 site was more reactive with aIIbb3 than the parent fibrinogen, suggesting that this binding site 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. Interactions forces >60 pN were more effectively reduced by EDTA than by any of the inhibitory peptides, indicating their dependence on the structural integrity and functionality of aIIbb3. The free γC dodecapeptide was less efficient than the cRGD peptides or integrilin in preventing the stronger interactions. Taken together, these data demonstrate that surface-bound fibrinogen and monomeric as well as polymeric fibrin are highly reactive with the aIIbb3. Fibrin is more reactive than fibrinogen in terms of binding probability and has higher binding strength. Fibrin-aIIbb3 binding is less sensitive to specific b3 integrin inhibitors, suggesting that fibrin and fibrinogen have distinct specificities towards aIIbb3. 



Additional Information: ISMCM

Open to: General Public
Admission: Free
Sponsor: Mathematics

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