Investigation of alternative mechanisms of collagen-induced platelet activation by using monoclonal antibodies to glycoprotein IIb-IIIa and fibrinogen.

Anti-HuPl-ml (reactive with IIIa) and anti-C2G7 (reactive with the carboxyl terminal of the alpha chain of fibrinogen) were used to investigate platelet aggregation, fibrinogen binding and thromboxane synthesis induced by low dose collagen (LDC) or high dose collagen (HDC) in normal or aspirin treated platelets. Anti-HuPl-ml and anti-C2G7 inhibited LDC induced platelet aggregation almost completely whilst abolishing fibrinogen binding; thromboxane production although reduced, still occurred. Thus LDC activation was dependent on fibrinogen binding to Gp IIIa. Aggregation of platelets induced by HDC was inhibited with anti-C2G7 or anti-HuPl-ml by 15-35% in association with a modest reduction in thromboxane (TxB2) production (with anti-HuPl-ml) and total inhibition of fibrinogen binding. When anti-HuPl-ml was added to aspirin treated platelets, aggregation with HDC although substantially reduced was not totally abolished. Collagen appears to activate platelets in a dose related manner in which there are at least three possible mechanisms via: (i) GpIIb-IIIa and associated fibrinogen binding; (ii) prostaglandin pathway; (iii) an alternate pathway responsible for approximately 20%-30% of platelet aggregation.

Changes in platelet function and reactivity induced by quinine in relation to quinine (drug) induced immune thrombocytopenia.

Quinine, a drug known to induce immune mediated thrombocytopenia, has been postulated to mediate binding of drug dependent antibodies to a range of platelet membrane glycoproteins. Quinine may not act solely as a hapten however, as we have shown that it inhibits platelet aggregation (ex vivo and in vitro) and release and modifies the ability of activated platelets to bind the adhesive proteins fibrinogen and fibronectin in a dose dependent fashion. Studies on the effect of quinine on the binding of monoclonal antibodies HuPlml (GpIIIa) FMC25 (GpIX) and AN51 (GpIb) to platelets shows a selective reduction in AN51 binding. In addition quinine induced platelet antibodies from thrombocytopenic patients, in the presence of quinine, have been shown to inhibit binding of these monoclonal antibodies to platelets to varying degrees. These observations suggest that quinine causes widespread but specific conformational changes in platelet membrane antigens which may expose neoantigens resulting in the production of quinine induced antibodies.

Estimation of platelet size by measurement of intracellular water space using an oil technique.

The intracellular water space of human platelets has been measured after equilibration with tritiated water and then separating these cells by centrifugation through phthalate oil of density 1.042. The mean intracellular water space of platelets in citrated plasma was 0.52 +/- 0.09 microliter/10(8) cells for 19 normal subjects. The gravimetric water content of platelets was 784 +/- 4 mg water/g cells. From these values the mean platelet volume was calculated to be 6.2 fl which agrees closely with values based on Coulter size distribution and thrombocytocrit. Gel filtration alters platelets such that a mean 19% of the platelets could not be centrifuged through phthalate oils of density 1.031 or 1.042. The measurement of tritiated water space of platelets centrifuged from their own plasma through oil provides a simple and reliable estimate of the mean platelet size.

Role of A alpha chain of fibrinogen in coagulation and platelet interaction investigated with a monoclonal antibody.

A murine monoclonal antibody (anti-C2G7), reactive with fibrinogen, was used to analyse the structure and function of the fibrinogen epitope C2G7. Anti-C2G7 was found to be reactive with fibrinogen but not with fibronectin, Factor VIII-von Willebrand Factor (FVIII-vWF), beta-thromboglobulin (beta TG), platelet factor 4 (PF4) nor with a range of normal cells and cell lines. Biochemical and plasmin digestion studies of fibrinogen revealed that C2G7 is present on the carboxy-terminal end of the alpha chain on a fragment with a Mr approximately 30-40 K. Functional studies, on the role of fibrinogen in coagulation and platelet function, demonstrated the importance of C2G7 (or a closely associated region) for thrombin-associated fibrin polymerization and collagen induced fibrinogen binding to platelets.

Plasma-dependent and -independent mechanisms of platelet aggregation induced by human tumour cell lines.

Tumour cell induced platelet aggregation (TCIPA) may facilitate haematogenous tumour metastasis. In this study of the aggregatory responses of human platelets to human tumour cell lines, we have found two distinct mechanisms of TCIPA. Colon carcinoma lines Colo 205 and Colo 397 produced TCIPA which was dependent upon thrombin generated through the activation of clotting factor VII, consistent with the expression of tissue factor activity by these cells. This mechanism was calcium dependent and was partially mediated by platelet ADP release as it was inhibited by apyrase. A uterine carcinosarcoma line (Colo 526) produced TCIPA by a novel mechanism which was dependent upon calcium, but was independent of thrombin generation and of the presence of plasma proteins, indicating that this aggregatory response is initiated by a direct platelet-tumour cell interaction.

The use of 125I labelled staphylococcal protein A in the diagnosis of autoimmune thrombocytopenic purpura and other immune mediated thrombocytopenias.

Platelet-associated immunoglobulin (IgG) has been measured directly on platelets from normal and thrombocytopenic subjects by a modified radioactive Coombs technique. Platelet-associated IgG was detected using 125I-staphylococcal protein A after which platelets were separated from unbound 125I-protein A by centrifugation through oil. Elevated values of platelet-associated IgG were observed in 17 of 19 patients with autoimmune thrombocytopenic purpura. All patients with thrombocytopenia associated with systemic lupus erythematosus or rheumatoid arthritis showed elevated platelet-associated IgG, while normal values were found in diverse thrombocytopenias which were non-immunological in origin. Measurement of anti-platelet antibody levels by an indirect test was less reliable in diagnosing autoimmune thrombocytopenic purpura, since only 7 of the 19 patients gave elevated values. However, the indirect test was reliable in the diagnosis of quinine-dependent immune thrombocytopenia; all 6 patients with suspected quinine purpura gave strongly positive indirect tests when 1 mM quinine was added to the patient's plasma. The use of 125I-protein A to detect platelet-associated IgG provides a rapid and simple technique for the reliable diagnosis of immune thrombocytopenia.

Characterization of platelet aggregation induced by the human carcinosarcoma Colo 526: role of platelet activation, tumor cell cytoskeleton and tumor cell plasma membrane.

Tumor cell-platelet interactions have been shown to be involved in the process of metastasis. This study characterizes the aggregation of washed platelets induced by the human uterine carcinosarcoma Colo 526. Ultrastructural studies revealed a two-stage process in which the earliest events were the adhesion and degranulation of individual platelets in contact with the tumor cell membrane. The second stage consisted of a wave of aggregation involving all residual platelets. We found that the first stage was initiated by a factor integral to the tumor cell plasma membrane which acted independently of the tumor cell cytoskeleton or metabolic processes. This factor was found to be a glycoprotein or glycolipid with functionally important sialic acid and N-linked carbohydrate residues. The initial stage was not dependent on platelet activation as neither aspirin nor prostacyclin prevented adhesion or degranulation. The second stage was found to be dependent on platelet activation. These results suggest that platelet aggregation induced by Colo 526 involves a distinctive primary stage which is initiated by a factor on the tumor cell plasma membrane resulting in the degranulation and lysis of individual platelets. This process can occur independently of platelet activation or aggregation and thus may have some relevance to the clinical use of platelet antagonists as antimetastatic agents.

The role of factor XI in the coagulant activity of platelets.

Washed platelets were ruptured by freezing and thawing; a coagulant activity was released which would correct the clotting time of factor XI (FXI)-deficient plasma only in the presence of kaolin. Platelets from a FXI-deficient patient treated in a similar fashion also released a coagulant activity which could be absorbed onto Sepharose-heparin and eluted similarly to plasma FXI. Collagen was employed to induce a coagulant activity in platelet-poor plasma (PPP) and platelet-rich plasma (PRP) in the presence and absence of antibodies developed to purified FXI and FXII. The presence of FXII antibody had little effect on the activity induced in PRP. However, the presence of FXI antibody eliminated the difference between PPP and PRP. An activity was induced when FXI-deficient PRP was incubated with collagen and none with PPP. One type of collagen failed to induce a coagulant activity.

The role of fibronectin in platelet aggregation.

A monoclonal antibody (anti-Fn2) was prepared which was reactive with both plasma fibronectin and fibronectin located within the platelet alpha granule. Immunoblotting analysis, on thermolysin digestion fragments of fibronectin, identified two immunoreactive fragments of Mr 145 kDa and 155 kDa which are known to contain a cell and DNA binding region. Anti-Fn2 was found to inhibit binding of fibronectin to platelets and DNA. Functional platelet studies, measuring platelet aggregation and 14C-serotonin release in washed platelet systems, demonstrated the ability of anti-Fn2 to totally inhibit low dose thrombin and low-dose collagen induced platelet aggregation and serotonin release. Anti-Fn2 partially inhibited platelet aggregation induced by ADP (10 microM) and arachidonic acid, but had no effect on platelet aggregation induced by high-dose thrombin or by the calcium ionophore A23187. These studies indicate that fibronectin participates in platelet aggregation and release induced by a range of agonists and suggest that it has a more important involvement in platelet function than previously described.

The role of platelet surface proteins reacting with heterologous antibodies.

Platelets were labeled with surface-labelling agents which identified up to six platelet membrane proteins. Three of these labelled proteins were recognised by a specific anti-platelet membrane serum by immunoprecipitation. Affinity fractionation of platelets with insolubilised IgG obtained from the anti-platelet membrane serum indicated a greater reaction between the antiserum and the platelet surface. Fab fragments from the antiserum inhibited ristocetin-induced aggregation of platelets and partially inhibited collagen-induced aggregation but did not affect ADP aggregation. Crossed immunoelectrophoresis of platelets using the anti-platelet membrane serum showed that the antiserum was specific for platelet membranes.

Detection of glycoprotein IIb and IIIa by monoclonal antibodies.

Murine monoclonal antibodies were produced against human platelet membranes and screened on platelets by a 125I protein A radioimmunoassay. Several clones produced platelet specific antibodies as they showed no reaction with peripheral blood lymphocytes, neutrophils, bone marrow (excluding megkaryocytes) or several cell lines. Two antibodies (designated anti-HuPl-m1a and anti-HuPl-m1b) were of particular interest in that although platelet specific they were non-reactive with platelets from a thrombasthenic patient. In functional assays these two antibodies could specifically inhibit ADP and collagen induced aggregation of platelets and release of ATP, retard platelet aggregation and ATP release induced by epinephrine, and inhibit ADP induced platelet fibrinogen binding. These two antibodies appear to recognize glycoproteins IIb and IIIa as analysis by SDS-PAGE using radiolabelled membranes revealed a two chain structure of molecular weight 112000 and 122000 daltons when run after reduction and 87000 and 140000 daltons non-reduced.