The Interaction of Cross-Linked Fibrin Fragment D-Dimer with αIIbβ3 Requires Receptor Activation, Occurs at Sites Other Than the γ-404-411 Sequence, and Contributes to Clot Retraction
Authors: Zafar Hina , Li Jihong , Buitrago Claudia , Nesic Dragana , Coller Barry S. 
Published: October 2019

Abstract
Background: The interaction between the fibrinogen (fbg) γ-chain sequence 404-411 and the 'RGD pocket' formed by αIIb and β3 plays a major role in platelet (P) aggregation. It requires activation of αIIbβ3 when fbg is in solution, but not when fbg is immobilized. Less is known about the interaction of Ps with cross-linked fibrin, which presumably is the dominant form of fibrin(ogen) in human thrombi and the form that participates in clot retraction. αIIbβ3 is required for clot retraction since it is decreased or absent in Glanzmann thrombasthenia patients who lack a functional receptor. Paradoxically, clot retraction is essentially normal with fbg lacking γ-408-411 or in the presence EDTA, which eliminates fbg binding to the RGD pocket. We recently studied the interaction of αIIbβ3 with fbg fragments D100 (which has γ-404-411) and 'D98' (which essentially lacks γ-406-411) to identify fbg binding sites on αIIbβ3 other than the RGD pocket. We found that: 1. activated, but not unactivated, Ps adhere well to immobilized 'D98;' 2. cells expressing normal αIIbβ3 could bind to: a) fbg in the absence of EDTA, but not in the presence of EDTA, b) D100 in the absence or presence of EDTA, c) 'D98' in the presence, but not the absence, of EDTA; 3. cells expressing constitutively active αIIbβ3 mutants, but not cells expressing normal αIIbβ3, adhere well to immobilized 'D98.' These data support there being two separate mechanisms of binding, one involving the interaction of γ-404-411 (in fbg and D100) with the αIIbβ3 RGD binding pocket, and another involving a site on D100 and 'D98' (but cryptic in fbg) that interacts with a site on αIIbβ3 exposed by receptor activation or induced by EDTA.
We have now studied adhesion of αIIbβ3 to the cross-linked fibrin fragment D-dimer (D-d). We predicted it to be similar to the interaction of 'D98' with αIIbβ3 because γ-K406, which is important in the binding of fbg to αIIb, participates in the antiparallel reciprocal factor XIIIa-mediated transglutaminase crosslinks between K406 and Q398 on two different γ-chains, and so is unlikely to be able to interact with the RGD pocket.
Methods: D-d, purified from cross-linked fibrin after plasmin digestion, was obtained from Haematologic Technologies and found to contain a dominant band with the expected Mr and immunoblot pattern using antibodies to the individual fbg chains. Fibrinogen, 'D98,' and HEK293 cells expressing normal and mutated αIIbβ3 were prepared as we previously described (Zafar et al., Blood Advances 2017), and the adhesion assays were conducted as described in that paper. Clot retraction was performed by adding BSA, 'D98,' or D-d (200 µg/ml) to washed Ps (2 X 108/ml) + thrombin (0.2 U/ml) in a glass aggregometer cuvette.
Results: αIIbβ3-mediated adhesion to immobilized D-d (10 µg/ml coating concentration) was similar to adhesion to 'D98' in that 1. Unactivated P adhered well to fbg, but poorly to 'D98' and D-d (n=4); 2. Activated Ps (10 µM thrombin receptor activating peptide) adhere ~2-fold better than unactivated Ps to fbg, but ~10-fold better to 'D98' and D-d (p<0.001 for unactivated vs activated Ps with both 'D98' and D-d; n=4); 3. HEK293 cells expressing normal αIIbβ3 (αIIbβ3-HEK) adhere well to fibrinogen, but not 'D98' and D-d; 4. HEK293 cells expressing the constitutively active aIIbF992A/F993Aβ3 receptor [αIIb(FF)β3-HEK] bound well to fbg, 'D98, and D-d (p<0.001 for both 'D98' and D-d compared to normal αIIbβ3; n=5); 5. EDTA treatment of Ps results in loss of adhesion to fbg, but significantly enhanced adhesion to 'D98' and D-d (p<0.05 for each ligand with and without EDTA; n=4); 6. EDTA treatment of αIIbβ3-HEK results in loss of adhesion to fbg, but significantly enhanced adhesion to both 'D98' and D-d (p<0.01 for each ligand with and without EDTA; n=5); 7. Adhesion of αIIb(FF)β3-HEK to D-d is like adhesion to 'D98' in being inhibitable by mAbs 7E3 and 10E5, eptifibatide, tirofiban, and RUC-4 (p<0.01 for all); n=4); 8. Adhesion of αIIb(FF)β3-HEK to D-d is similar to adhesion to 'D98' in being inhibitable by mutating β3 D119 to A in the MIDAS (p<0.01; n=6); 9. EDTA-induced adhesion of HEK-αIIbβ3 to both 'D98' and D-d is not inhibitable by mutating β3 D119 to A. 10. 'D98' and D-d inhibited clot retraction, but BSA did not.
Conclusion: We conclude that cross-linked fibrin fragment D-dimer, like fibrinogen fragment 'D98', interacts with activated αIIbβ3 via sites other than the γ-404-411 peptide, and that this interaction contributes to clot retraction.

Disclosures
Coller: Rockefeller University: Patents & Royalties: RUC-4; Platelet Biogenesis: Consultancy; Centocor: Patents & Royalties: Abciximab; CeleCor: Consultancy, Equity Ownership; Accumetrics: Patents & Royalties: VerifyNow Assays; Scholar Rock: Consultancy, Equity Ownership.