Cycles of Agglutination‐Disagglutination Induced by Ristocetin in Thrombasthenic Platelets

769

274

A B S T R A C T To define better the role of the fibrinogen receptor in platelet physiology and to characterize it biochemically, a murine monoclonal antibody that completely blocks the binding of fibrinogen to the platelet surface was produced by the hybridoma technique with the aid of a functional screening assay. Purified F(ab')2 fragments and/or intact antibody completely blocked aggregation induced by ADP, thrombin, or epinephrine and the binding of radiolabeled fibrinogen to platelets induced by ADP. The antibody did not block agglutination of formaldehyde-fixed platelets by ristocetin or shape change induced by either ADP or thrombin. ADP-and epinephrine-induced release of ATP was completely inhibited by the antibody, but inhibition of release induced by collagen and thrombin was dose dependent and partial. The antibody also dramatically inhibited platelet retention in glass-bead columns, platelet adhesion to glass, and clot retraction. Thus, the antibody induced a thrombasthenic-like state. Immunofluorescent studies confirmed the specificity of the antibody for normal platelets and megakaryocytes and suggested that there is a marked decrease in detectable antigen in thrombasthenic platelets. Radiolabeled antibody bound to an average of -40,000 sites on normal platelets but it bound to <2,000 sites on the platelets of a patient with thrombasthenia. The antibody immunoprecipitated both glycoproteins IIb and IlIa, and both glycoproteins bound to an affinity column of the antibody. These Presented in part to the American Society of Hematology, San Antonio, TX, December 1981, and published in abstract form in Blood, 1981, 58:191a.

Section: Discussionsupporting

confidence: 79%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

A murine monoclonal antibody that completely blocks the binding of fibrinogen to platelets produces a thrombasthenic-like state in normal platelets and binds to glycoproteins IIb and/or IIIa.

Coller¹,

Peerschke²,

Scudder³

et al. 1983

769

274

“…Most reports indicate that thrombasthenic platelets do not aggregate at all in response to collagen (21). However, studies by several investigators showed that small aggregates are, in fact, produced (43)(44)(45)(46). Similar results are also obtained after in vitro addition to normal platelets of a monoclonal antibody to GPIIb-IIIa complex (19).…”

supporting

confidence: 74%

A myeloma paraprotein with specificity for platelet glycoprotein IIIa in a patient with a fatal bleeding disorder.

DiMinno¹,

Coraggio²,

Cerbone³

et al. 1986

Impaired platelet aggregation, normal shape change, and agglutination and normal ATP secretion and thromboxane synthesis in response to high concentrations of thrombin or arachidonic acid were found in a patient with multiple myeloma and hemorrhagic tendency. The purified IgG1 kappa or its F(ab'}2 fragments induced similar changes when added in vitro to plateletrich plasma from normal subjects. In addition, the paraprotein inhibited adhesion to glass microbeads, fibrin clot retraction, and binding of radiolabeled fibrinogen or von Willebrand factor to platelets exposed to thrombin or arachidonic acid without affecting intraplatelet levels of cAMP. The radiolabeled paraprotein bound to an average of 35,000 sites on normal platelets but it bound to <2,000 sites on the platelets from a patient with Glanzmann's thrombasthenia. Immunoprecipitation studies showed that the platelet antigen identified by the paraprotein was the glycoprotein IlIa. Furthermore, binding of radiolabeled prostaglandin El (PGEI) to resting platelets as well as binding of von Willebrand factor to platelets stimulated with ristocetin were entirely normal in the presence of patient's inhibitor. These studies indicate that bleeding occurring in dysproteinemia may be the result of a specific interaction of monoclonal paraproteins with platelets. In addition, our data support the concept that the interaction of fibrinogen and/or von Willebrand factor with the platelet glycoprotein Ilb-IIla complex is essential for effective hemostasis.

“…The initial response of platelets to relatively high concentrations of ristocetin is normal in both afibrinogenemia (21) and Glanzmann thrombasthenia (30), since a different platelet receptor, namely GPIb, and a different adhesive glycoprotein, namely vWF, are involved in this response (10). At lower ristocetin concentrations, however, aggregation may be abnormal in both conditions (21,30). This finding may reflect the participation of fibrinogen in platelet aggregation mediated by vWF binding to GPIb, as suggested by recent studies (31,32).…”

Section: Discussionmentioning

confidence: 99%

von Willebrand factor interaction with the glycoprotein IIb/IIa complex. Its role in platelet function as demonstrated in patients with congenital afibrinogenemia.

Marco¹,

Girolami²,

Zimmerman³

et al. 1986

176

We have studied three afibrinogenemic patients, who had only trace amounts of plasma and platelet fibrinogen as measured by radioimmunoassay, and demonstrate here that the residual aggregation observed in their platelet-rich plasma is dependent upon von Willebrand factor (vWF) binding to the platelet membrane glycoprotein (GP)IIb/IIIa complex. The abnormality of aggregation was more pronounced when ADP, rather than thrombin, collagen, or the combination of ADP plus adrenaline was used to stimulate platelets. With all stimuli, nevertheless, the platelet response was completely inhibited by a monoclonal antibody (LJP5) that is known to block vWF, but not fibrinogen binding to GPIIb/IIIa. Addition of purified vWF to the afibrinogenemic plasma resulted in marked increase in the rate and extent ofaggregation, particularly when platelets were stimulated with ADP. This response was also completely blocked by LJP5.Addition of fibrinogen, however, restored normal aggregation even in the presence of IPS, a finding consistent with the knowledge that antibody LJP5 has no effect on platelet aggregation mediated by fibrinogen binding to GPIIb/IIIa. Two patients gave their informed consent to receiving infusion of 1-desamino--D-arginine vasopressin (DDAVP), a vasopressin analogue known to raise the vWF levels in plasma by two-to fourfold. The bleeding time, measured before and 45 min after infusion, shortened from >24 min to 12 min and 50 s in one patient and from 16 min to 9 min and 30 s in the other. Concurrently, the rate and extent of ADP-induced platelet aggregation improved after DDAVP infusion. The pattern, however, reversed to baseline levels within 4 h. The concentration of plasma vWF increased after DDAVP infusion, but that of fibrinogen remained at trace levels. We conclude that vWF interaction with GPIIb/Il~a mediates platelet-platelet interaction and may play a role in primary hemostasis.