Characterization of the gamma chain platelet binding site on fibrinogen fragment D

Abstract

Glycoprotein (GP) IIb/IIIa on adenosine diphosphate (ADP)-activated human platelets interacts with specific sites on the fibrinogen molecule leading to aggregation. We characterized the platelet-binding site on the gamma chains of fibrinogen using plasmic fragments D gamma A and D gamma'. Fragment D gamma A, which contains the carboxy terminal gamma A400–411 platelet-binding sequence (HHLGGAKQAGDV), was 70-fold more active than the synthetic gamma A400–411 peptide in inhibiting ADP- induced platelet aggregation. Fragment D gamma A inhibited fibrinogen binding and also bound directly to ADP-activated platelets. The Kd values determined for fibrinogen and fragment D gamma A binding were 0.55 mumol/L and 1.2 mumol/L, respectively. In contrast, fragment D gamma', which differs from fragment D gamma A with respect to its gamma chain sequence from position 408 to the COOH-terminus at position 427, did not inhibit platelet aggregation or fibrinogen binding, and did not bind directly to the platelet surface. Denaturation of fragment D gamma A with guanidine-HCl caused a loss of inhibitory activity in platelet aggregation assays. These data indicate that the native conformation of the gamma chain platelet-binding site on fibrinogen is important for optimal binding to GPIIb/IIIa.

Thrombosis risk modification in transgenic mice containing the human fibrinogen thrombin-binding γ′ chain sequence

“…1). Read‐through into the hu‐intron 9 region is predicted to produce a chimeric murine‐human γ′ chain (hu‐γ′) that: (i) retains the mu‐γ chain 398/399 glutamines at and lysine 406 that become cross‐linked by FXIII; (ii) retains the FXIII B subunit binding site on its γ′ chains that entrain the A subunits of the FXIII A 2 B 2 complex; (iii) possesses the hu‐γ′ chain thrombin‐binding sequence [4,6,8,29,40]; and (iv) lacks a platelet interactive site that binds to αIIbβ 3 ‐integrin receptors [41]. The mutated γ′‐gene contained a neomycin resistance cassette flanked by lox P sites.…”

Identification and Characterization of the Thrombin Binding Sites on Fibrin

“…The rat and human ␥Ј chains are tyrosine-sulfated at ␥Ј418 (31,32) and also at ␥Ј422 in humans. 2 ␥ A and ␥Ј chains are functionally equivalent with respect to factor XIIIa-catalyzed cross-linking (23), but unlike the ␥ A chain, ␥Ј chains lack the complete platelet binding sequence, ␥ A 400 -411, and therefore do not support ADP-induced fibrinogen binding or platelet aggregation (33)(34)(35). Our group has recently presented evidence that plasma factor XIII binds specifically to ␥Ј chains (36), but little else is known about its functions.…”

Inherited Dysfibrinogenemia: Emerging Abnormal Structure Associations With Pathologic and Nonpathologic Dysfunctions

“…A minor fraction of circulating fibrinogen contains chains that differ in that amino acids at positions 408 to 411 are replaced by a unique 20 amino acid sequence extension. 48 This different chain arises from differential mRNA splicing or processing of the primary chain mRNA transcript. Molecules containing such chains are functionally normal with respect to coagulability and to their capacity to be cross-linked by Factor XIIIa, but fail to support adenosine diphosphate (ADP)-induced platelet aggregation and to bind to platelets.…”