Activation of human platelets by a stimulatory monoclonal antibody.

The clinical significance of the interaction of antibodies with circulating platelets is well documented, but the mechanisms underlying these interactions are not fully known. Here we describe the characterization of anti-human platelet membrane protein monoclonal antibody (mAb) termed F11. Interaction of mAb F11 with human platelets resulted in dose-dependent granular secretion, measured by [14C]serotonin and ATP release, fibrinogen binding and aggregation. Analysis of the specific binding of mAb F11 to platelets revealed a high affinity site with 8,067 +/- 1,307 sites per platelet with a dissociation constant (Kd) of 2.7 +/- 0.9 x 10(-8) M. Two membrane proteins of 32,000 and 35,000 daltons, identified by Western blotting, were recognized by mAb F11. Incubation of 32Pi-labeled platelets with mAb F11 resulted in rapid phosphorylation of intracellular 40,000- and 20,000-dalton proteins, followed by dephosphorylation of these proteins. Monovalent Fab fragments or Fc fragments of mAb F11 IgG did not induce platelet aggregation or secretion; however, Fab fragments of mAb F11 IgG blocked mAb F11-induced platelet aggregation and the binding of 125I-mAb F11 to platelets. The addition of an anti-GPIIIa monoclonal antibody (mAb G10), which inhibits 125I-fibrinogen binding and platelet aggregation, completely blocked mAb F11-induced [14C]serotonin secretion and aggregation but not the binding of 125I-mAb F11 to platelets. mAb G10 also inhibited the increase in the phosphorylation of the 40,000- and 20,000-dalton proteins induced by mAb F11. These results implicate the involvement of the GPIIIa molecule in the chain of biochemical events involved in the induction of granular secretion.

The clinical significance of the interaction of antibodies with circulating platelets is well documented, but the mechanisms underlying these interactions are not fully known.
Here The addition of an anti-GPIIIa monoclonal antibody (mAb GlO), which inhibits "'I-fibrinogen binding and platelet aggregation, completely blocked mAb Fll-induced ['"Clserotonin secretion and aggregation but not the binding of '261-mAb F 11 to platelets. mAb GlO also inhibited the increase in the phosphorylation of the 40,000-and 20,000-dalton proteins induced by mAb Fl 1, These results implicate the involvement of the GPIIIa molecule in the chain of biochemical events involved in the induction of granular secretion.
Platelets and platelet membrane glycoproteins play a significant role in immunologic reactions. Early studies have suggested that alloantibodies developed in patients following multiple transfusions activate platelets in vivo resulting in thrombocytopenia (1,2). Specific anti-platelet autoantibodies and alloantibodies to membrane glycoproteins (GP)' such as GPIb, GPIIb, GPIIIa, and GPV now have been identified in patients with clinical disorders of drug-dependent thrombo- cytopenia purpura, posttransfusion purpura, neonatal isoimmune thrombocytopenia, chronic immune thrombocytopenia purpura, and septicemia (3-16). AIDS patients with acute thrombocytopenia purpura were shown to have anti-platelet antibodies (17,18). The study of the interaction of immunoglobulins with platelets has been enhanced by the development of monoclonal antibodies which induce platelet aggregation (19)(20)(21)(22)(23)(24)(25)(26). The study of such monoclonal antibodies enables the identification of specific platelet membrane antigens involved in platelet activation by immunoglobulins in vivo, and in the elucidation of the molecular mechanisms resulting in this activation process.
In this paper we describe the properties and mechanism of action of a novel monoclonal antibody which acts as a potent inducer of aggregation and secretion in human platelets. This monoclonal antibody recognizes a unique receptor on the platelet surface which is involved in platelet activation, and we present data showing the association of the fibrinogen receptor in platelet secretion.

S.D.) of [Y]5HT
release in three separate experiments performed in triplicate.
Similar results were obtained with mAb Fll added to platelet-rich plasma. Washed platelets, pretreated with chymotrypsin (30), also responded to mAb Fll by secreting ATP and aggregating in the absence of exogenously added fibrinogen.
Similar secretion and aggregation were observed with platelets pretreated with human granulocyte and pancreatic elastase.
As shown in Fig. 1, mAb Fll-induced platelet aggregation (top panel) and ATP release (bottom panel) were not immediate events but were initiated after a latency period which was dependent on the concentration of mAb Fll. The latency observed for platelet activation following the addition of mAb Fll was shortened with increasing concentrations of mAb Fll. Approximately 5.8 pg/ml of mAb Fll-induced platelet activation within 3 min, whereas with higher concentrations of mAb Fll the latency period decreased to less than a minute.
Binding of '251-mAb Fll to Platelets-The binding of "'I-mAb Fll to platelets increased rapidly with time and reached equilibrium within 15 min. The binding of mAb Fll to platelets was dependent on the concentration of radiolabeled ligand. Fig. 2  the data from all 11 experiments indicated a single class of binding sites with 8067 f 1307 binding sites/platelet with a dissociation constant of 2.7 + 0.9 X 10eR M. These values are the weighted means (x f SE.) which were calculated by using the correlation coefficient as a weighting factor. Stimulation of Protein Phosphorylation in Platelets by mAb FII-The effect of mAb Fll on the phosphorylation of intracellular platelet proteins is shown in Fig. 3. Following incubation with mAb Fll, we found a selective and time-dependent increase in the phosphorylation of proteins with apparent molecular weights of 40,000 and 20,000. Such changes in phosphorylation pattern were found in intact platelets and in chymotrypsin-pretreated platelets. The phosphorylation of the 40,000-and 20,000-dalton proteins increased significantly within seconds following the addition of mAb Fll. In intact platelets, the maximal increase in the phosphorylation of these proteins occurred following 5 min of incubation with mAb Fll. After longer incubations there was a decrease in the phosphorylation state of both the 40,000-and 20,000dalton proteins. The changes in the phosphorylation state of the 40,000-and 20,000-dalton proteins induced by mAb Fll in platelets pretreated with chymotrypsin followed essentially the same time course shown for intact platelets in Fig. 3.
Platelet Proteins Recognized by mAb Fll-The platelet proteins recognized by mAb Fll in a Western immunoblotting procedure are shown in Fig. 4. mAb Fll recognized epitopes on two platelet-membrane proteins with molecular masses of 32 and 35 kDa. Both of these proteins were recognized by mAb Fll in 18 separate experiments conducted to date. Another monoclonal antibody, named mAb GlO, which is described below, as well as immunoglobulins obtained from SpB/O-injected mice (Fig. 4), showed no interaction with these proteins.
Involvement of GPIIb-IIIa in the Activation of Intact Platelets by mAb F-l I-We isolated a second monoclonal antibody, termed GlO, which is directed against the platelet GPIIIa molecule. Fig. 5 shows the results of Western blotting experiments using three different antibodies for comparison. mAb GlO is shown to immunoblot GPIIIa (lane A). As previously shown by us (30) The mAb Fll-induced platelet aggregation, ATP release, ['C]5HT secretion and protein phosphorylation were tested in the presence of the anti-glycoprotein IIIa monoclonal antibody mAb GlO. mAb G10 completely inhibited mAb Fllinduced platelet aggregation. Fifty percent inhibition of platelet aggregation and ATP release occurred at concentrations of mAb GlO ranging from 0.35 to 0.45 pg/ml (Fig 6A). A slightly higher concentration of mAb GlO (1.8 pg/ml) inhibited 50% of the mAb Fll-induced [W]5HT release. Fig. 6B shows that the stimulation by mAb Fll of the phosphorylation of the intracellular 40,000-and 20,000-dalton proteins by mAb F-11 was completely inhibited by mAb GlO. Fig. 6B also shows that the mAb Fll-induced increase in phosphorylation starts before aggregation (compare time points marked d in upper and lower panels of Fig. 6B). These results were not due to blockade by mAb GlO of mAb Fll binding to platelets, as described below.

Inhibition of mAb Fl l-induced Platelet Aggregation by mAb
Fll Fab Fragments-Fab fragments were prepared from purified IgG of mAb Fll. Neither these monovalent molecules nor Fc fragments induced granular secretion or platelet aggregation. The effects of monovalent Fab fragments on mAb Fll-induced platelet aggregation are shown in Fig. 7A. The addition of increasing concentrations of Fab fragments prolonged the latency of mAb Fll-induced platelet aggregation from 2 min to 1 h and longer. The mechanism responsible was found to be the inhibition by the Fab fragments of mAb Fll binding, as shown in Fig. 7B. The I&, of Fab fragment inhibition of mAb Fll binding is approximately 5 pg/ml. On the other hand, Fc fragments prepared from mAb Fll-IgG had no effect on mAb Fll binding to platelets, and did not inhibit mAb FIl-induced platelet aggregation even at a concentration as high as 435 rg/ml. In five separate experiments, mAb GlO-IgG did not inhibit the binding of ""I-mAb Fll to platelets. The binding data was similar to that seen in Fig.   7B  mAb Fl l-induced Aggregation in Chymotrypsin-pretreated Platelets- Fig.   8, A and B, shows the spontaneous aggregation of chymotrypsin-treated platelets upon the addition of fibrinogen in the presence and absence of PGEI. The result indicates that the elevation of cyclic AMP does not interfere with this type of aggregation. In contrast, the mAb Fll-induced aggregation of chymotrypsin-treated platelets is completely inhibited by PGE, as shown in Fig. 8  Fll IgG binding by mAb Fll Fab fragments. A, inhibition of platelet aggregation. mAb Fll (5 &g/ml)-induced platelet aggregation was performed in the presence of mAb Fll Fc fragments (90 rg/ml) or mAb Fll Fab fragments (90 @g/ml) as shown above. mAb Fll Fab and Fc fragments were incubated with platelets for 1 min at 37 "C prior to the addition of mAb Fll. mAb Fll Fc fragments did not inhibit mAb Fll-induced platelet aggregation. B, dose-dependent inhibition of 'Y-mAb Fll binding to platelets by mAb Fll Fab fragments. mAb Fll Fab (circles) or mAb Fll Fc fragments (triangles) were incubated with platelets for 1 min prior to the addition of lz51-mAb Fll. The binding of 'Z51-mAb Fll IgG to platelets was performed as described under "Experimental Procedures." Each point is the mean of at least two separate experiments. mAb G10 IgG gave results in at least five separate experiments which were similar to those observed with mAb Fll Fc fragments (triangles). ATP, and ATP analogues. Table I shows the I& values for  such inhibition by ATP, 5'-p-fluorosulfonylbenzoyladenosine, and AMP-PNP.
The simultaneous secretion of [14C]5HT from platelet-dense granules following the addition of mAb Fll was also measured (Fig. 9). We found that although ATP inhibited [14C]5HT secretion induced by mAb Fll (Fig. 9), maximal inhibition of secretion, even at high ATP concentra-  This would indicate that mAb Fll acts directly on the platelet surface to induce 30% granular secretion. Is the ADP receptor involved in this initial action? To test this possibility, platelets were made refractory to ADP by adding nanomolar concentrations of ADP as shown in the bottom panel of Fig. 10. Platelets which were made refractory to ADP still responded to mAb Fll with a shortened latency, even though maximal concentrations of ADP could not induce aggregation. This result, indicates that mAb Fll does not interact directly with the ADP receptor site. DISCUSSION We report here the characteristics and mechanisms of action of a monoclonal antibody named mAb Fll, a potent platelet agonist.. mAb Fll directly stimulates platelet secretion, measured as ATP and serotonin release, and fibrinogendependent platelet aggregation.
By interacting with a unique receptor, termed Fll, mAb Fll induces rapid intracellular phosphorylation of two major proteins: a 40,000-dalton protein which is a known substrate for protein kinase C, and a 20,000-dalton protein, the light chain of myosin and the substrate for myosin light, chain kinase, a Ca2+-dependent enzyme. Thus, the cascade of intracellular biochemical events triggered by mAb Fll involves stimulation of protein kinase C and elevation of free calcium ion levels, in all likelihood through activation of the phosphoinositide cycle (43). We have found that mAb Fll recognizes platelet surface membrane proteins of approximately 32,000 and 35,000 daltons, as determined by Western blotting and by analysis of the bound material eluted from a mAb Fll affinity column. By Scatchard analysis we have shown that there are approximately 8,000 high affinity Fll binding sites per platelet. The platelet Fll antigen appears to be resistant to surface proteolysis since we observed that chymotrypsin-and elastasepretreated platelets are stimulated by mAb Fll to secrete and aggregate. These proteolytically treated platelets show significant increases in intracellular phosphorylation of the 20,000- viously not recognized by other stimulatory antibodies.
Detailed characterization of the structure of this unique receptor and its associated glycoproteins is in progress. The fibrinongen receptor, consisting of glycoproteins IIb-IIIa, appears to play an important role in the action of mAb Fll.
A monoclonal antibody developed in our laboratory (named mAb GlO), which blocks aggregation and '*"I-fibrinogen binding to ADP-stimulated platelets, was found to be directed against GPIIIa. mAb GLO potently and completely inhibited mAb Fll-stimulated platelet aggregation. Moreover, mAb GlO blocks intracellular events induced by mAb Fll: these events include the increase in the phosphorylation of 40,000-and 26,600-dalton proteins and the initiation of ["Cl 5HT and ATP secretion. The complete inhibition by mAb GlO of intracellular protein phosphorylation events and the secretion induced by mAb Fll indicates that the GPIIIa molecule functions not only as a fibrinogen binding site required for fibrinogen-dependent platelet aggregation, but that GPIIIa also plays an important role in the transmission of signals that activate second messenger-generating systems leading to secretion. These results describe a new role for GPIIIa in platelet function.
In a previous report we have described the platelets of a Friedreich's ataxia patient with unique thrombopathy (44). mAb Fll induced secretion in platelets of this patient but not aggregation, due to a defect in the exposure of fibrinogen receptors. Interestingly, also in this patient mAb GlO inhibited mAb Fll-induced secretion, indicating that the role of GPIIIa in signals that lead to secretion can be separated from its role in exposure of fibrinogen binding sites.
In addition to mAb GlO, the Fab fragments of mAb Fll also inhibited the activation of platelets by mAb Fll. This inhibition was found to be due to direct interference of the Fab fragments with the binding of the mAb Fll IgG molecule to the platelet surface. Such interference is consistent with the possibility that mAb Fll-induced platelet activation involves receptor dimerization and microclustering (45) or the platelet Fc receptor (46). Agents which increase the level of cyclic AMP also inhibit mAb Fll-induced platelet aggregation, and this may be due to the inhibition of fibrinogen receptor exposure (47). The involvement of released ADP in mAb Fll-induced platelet aggregation is indicated by the finding that ATP and ATP analogues, which block the ADP receptor, and apyrase, which degrades the released ADP, completely inhibit aggregation.
However, significant granular secretion (30% of uptake) still occurs in response to mAb Fll even in the presence of either ATP or apyrase, indicating that the direct interaction of mAb Fll with its receptor results, in part, in granular release.
In conclusion, mAb Fll interacts with specific protein components (32 and 35 kDa) at the platelet surface. This interaction leads to platelet granular secretion and aggregation. The biochemical pathways of platelet activation by mAb Fll involve stimulation of the activity of protein kinase C and of the Ca'+/calmodulin-dependent myosin light chain kinase, and is inhibited by elevating intracellular cyclic AMP. The mAb Fll-induced aggregation of platelets appears to be secondary to ADP release. The mAb Fll-induced secretion appears to involve action of glycoprotein IIIa. At least 30% of the granular secretion induced by mAb Fll is not mediated by ADP but by a specific Fll receptor. Detailed characterization of this unique receptor will provide novel information on the process of platelet activation.