The platelet‐fibrinogen interaction

Abstract

Fibrinogen participates in platelet aggregation via specific inducible receptors on the cell surface. We have used a photoactivable bifunctional reagent, N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate, SANAH, to derivatize '2s1-labeled-fibrinogen (I2'1-Fg) and crosslink it to ADP-stimulated platelets. Binding experiments established that 1251-Fg and Iz5I-Fg-SANAH interacted with platelets with the same kinetics and affinity as unlabeled fibrinogen. After photoactivation of the platelet-bound '251-Fg-SANAH, polyacrylamide gel electrophoresis under reducing conditions revealed formation of a high molecular weight covalent complex with coordinate loss of the A a chain. I2'I-Fg-SANAH missing the extreme carboxy-terminal region of the Aa chain failed to crosslink to the platelets under similar conditions. Crosslinked "'1-Fg-SANAH was extracted from the cells in 1 % Triton X-100, and immunoprecipitation with antibodies specific for platelet membrane glycoproteins was used to identify components of the complex. With antibodies to the glycoprotein IIb/III complex (anti-GP IIb/III), 40 9 % of the extracted "'I-Fg-SANAH was irnmunoprecipitated. Omission of photoactivation, platelets, or ADP from the reaction or use of unmodified lZ5I-Fg resulted in less than 5 % immunoprecipitation by the anti-GP IIb/III. As controls for specificity, anti-(glycoprotein Ib) or anti-IgG immunoprecipitated less than 5 % of the extracted I2%Fg-SANAH. Under similar conditions, 45 "/, of the GP lTb/III from surface-labeled platelets was recovered in the antiGPITb/IIT immunoprecipitate. These results indicate that the Aa chain of fibrinogen comes in close proximity to GP IIb/III when the molecule is bound to its platelet receptor.Stimulated platelets interact with one another to form aggregates, a process essential to primary hemostasis. Fibrinogen may participate in this reaction by interacting with specific inducible rceptors on the platelet surface [l, 21, and receptor occupancy is closely related to the aggregation response of the platelets [3, 41. Recent studies have implicated two of the surface membrane glycoproteins, designated GP 1Ib and GF' 111, in platelet aggregation and fibrinogen receptor function. These two glycoproteins have apparent molecular weights of 136000 (GP IIb) and 95000 (GPIII) on polyacrylamide gel electrophoresis under n onreducing conditions [5] and form a noncovalent complex, GP Ilb,/III, in the presence of calcium [6-81. Thrombasthenic platelets, which generally lack both GP IIb and GP 111 [9, 201, do not aggregate in response to most platelet stimuli [Ill and fail to express the majority of fibrinogen binding sites [l].Other observations which also support a role for GP lIb/III in fibrinogen receptor function include: (a) an 1gG alloantibody directed against the G P IIb/III inhibits fibrinogen binding to platelets [12]; (b) partially purified GP IIb/III associates with fibrinogen in the presence of calcium in a solid __-_Ahbrevhtiunst SANAH, Succinimidyl-6-(4'-a~ido-2'-nitrophenylamino) hexanoate; lZ5I-...

Specific binding of human fibrinogen to cultured human fibroblasts

“…This indicates that structural loci, which are recognized by the membrane of the fibroblast are present in the N-terminal domain of the molecule. This contrasts with recent observations that the sites of interaction between fibrinogen and platelets are localized in the C-terminal part of the Aa and the y chains [12,21,221 and suggests that the binding of fibrinogen to fibroblasts is different from binding to platelets and that specific domains may exist in the molecule for the interaction of fibrinogen with different cellular systems.…”

Retention of the glycoprotein IIb-IIIa complex in the isolated platelet cytoskeleton. Effects of separable assembly of platelet pseudopodal and contractile cytoskeletons.

“…The molecular interactions in the platelet surface membrane which allow expression of the fibrinogen binding site following activation are currently unknown. Several lines of evidence identify the glycoprotein complex Ilb-IIMa found in the platelet surface membrane as the fibrinogen receptor (4,(7)(8)(9)(10)(11).…”

Divalent cation regulation of the surface orientation of platelet membrane glycoprotein IIb. Correlation with fibrinogen binding function and definition of a novel variant of Glanzmann's thrombasthenia.

“…Bound proteins were eluted in 0.1 M alpha methyl mannoside in the same buffer, and radioiodinated in detergent utilizing chloramine and 125I (2). The radioiodinated material was then immunoprecipitated by use ofa goat polyclonal anti-GPIIb-IIIa and protein A containing staphylococci exactly as described previously (12). The radiolabeled immunoprecipitate was eluted in sample buffer (28) and analyzed by SDS-PAGE under reducing conditions, and the major bands ofmol wt 123,000 (GPIIb alpha) and 105,000 (GPIII) were cut out, trypsin was digested, and the resultant peptides mapped by high-voltage electrophoresis followed by thin-layer chromatography exactly as described by Elder et al (29).…”