No abstract

SummaryA new monoclonal antibody (mAb), VM64, reacts with a common antigen on the surface of human platelets and vascular endothelial cells (EC). Under nonreduced conditions it recognized in immunoblotting a protein of 130 kDa both in platelets and EC. VM64 precipitated the same 130 kDa protein from the lysate of surface radioiodinated platelets. Electrophoretic mobility of this protein was not altered by reduction and differed from the bands precipitated by reference mAb against platelet glycoproteins (GP) Ia–IIa, Ib, IIb–IIIa and GMP130. VM64 binding to platelets and EC was specific and saturable. The number of binding sites on platelets was 9.9 ± 3.5 × 103 per platelet and on the surface of EC monolayer – 2.40 ± 0.32 × 106 per cell. VM64 also binds to platelets from Glanzmann's thrombasthenia patients which lack GPIIb–IIIa. VM64 did not affect platelet aggregation induced by ADP, collagen, thrombin and ristocetin. In the monolayers of EC from umbilical vein and human aorta, VM64 stained the area at the periphery of the cells adjacent to the cell-cell boundaries. In preconfluent cultures preferential staining was observed at the active leading margins of the cells. Unlike EC cultures from umbilical vein, where all cells were positively stained, in aortic EC cultures some unstained or poorly stained cells were constantly present, indicating a heterogeneity of EC population related to the expression of VM64 antigen. The biochemical characteristics of VM64 antigen, its presence both on platelets and EC and typical distribution on the surface of EC suggested that this antigen is identical to PECAM (CD31) protein.

Glycoproteins (GPs) IIb and IIIa form a Ca(2+)-dependent complex in platelet membrane and change their conformation upon platelet activation and dissociation of the complex. A new anti-GPIIIa monoclonal antibody (mAb), CRC54, is described which could distinguish different conformational states of GPIIIa. This antibody (i) precipitated GPIIb-IIIa from platelet Triton X-100-lysate, (ii) recognized the GPIIIa band in Western blotting of platelet SDS-lysate, and (iii) did not react with platelets from a Glanzmann's thrombasthenia patient lacking GPIIb-IIIa. Immunoblotting of chymotryptic digestion products of purified GPIIb-IIIa has shown that CRC54 epitope is located within residues 1-100 at the N-terminus of GPIIIa. CRC54 bound weakly to platelets in the presence of Ca2+ and Mg2+, 2.34 +/- 0.28 x 10(3) molecules per platelet at saturation. The same level of binding was observed without any divalent cations in the medium. However, binding of CRC54 was increased by several times after treatment of platelets with EDTA, 10.04 +/- 0.28 x 10(3) molecules per platelet. Increase of CRC54 binding correlated with the dissociation of GPIIb-IIIa complex which was followed by the decrease of the binding of another mAb, CRC64, directed against complex-specific epitope of GPIIb-IIIa. Binding of CRC54 to platelets was changed neither by platelet activation in suspension with thrombin or ADP nor by the occupancy of GPIIb-IIIa ligand binding site with GRGDSR peptide. However, binding was significantly stimulated by platelet adhesion to polystyrene plastic. As measured using 51Cr-labelled platelets, binding of 125I-CRC54 to adherent platelets in the presence of divalent cations was about 4 times higher than to platelets in suspension, 8.68 +/- 0.48 x 10(3) per platelet. This increase was not due to the dissociation of GPIIb-IIIa since complex-specific antibody CRC64 still bound effectively to the surface of adherent platelets. The data obtained indicated that: (1) CRC54 recognized an epitope specific for the dissociated form of GPIIIa; (2) the CRC54-reactive epitope of GPIIIa is also expressed on the surface of adherent platelets.

Platelet activation induced by monoclonal antibodies (mAB) was studied using three stimulatory mAB (all IgG(1)) against different platelet surface glycoproteins: VM58 against GPIV, LeoAl against PTA1, and FMC 56 against CD9. F(ab')(2) fragments of these antibodies failed to activate platelets themselves but blocked platelet aggregation induced by the relevant intact antibody. Platelet aggregation was also completely blocked by the anti-FcγRII (Fc-receptor) monoclonal antibody, IV.3. A heterogeneity of platelet response to stimulatory mAB was observed amongst normal donors. All three antibodies added to platelet-rich plasma (PRP) from responders induced full platelet aggregation and dense body release. However, in PRP from nonresponders, VM58 and LeoAl did not induce platelet activation whilst FMC 56 activated platelets but to a lesser extent than in responders (longer lag phase and reduced release). The ratio of responders to nonresponders was ∼ 1:1 (n = 110). The heterogeneity was not due to differences in the copy number of either the antigen (VM58) or FcγRII. The ability of donor platelets to be aggregated by stimulatory mAB in PRP correlated with the ability of these platelets to respond to aggregated murine IgG(1) (mAB irrelevant to platelets). The combined results suggest that both the Fab and Fc region of stimulatory mAB are necessary in order to induce a platelet response and that this response is mediated through FcγRII. The difference between responders and nonresponders can be explained by the known polymorphism of FcγRII (Looney et al, 1988) and the capacity of the polymorphic forms of FcγRII to bind and to respond to murine IgG(1).

In this study, we have examined whether the platelet Fc-receptor, FcγRII (CD32), is associated with either of the two major platelet membrane glycoproteins, the GPIb-IX complex and the GPIIb-IIIa complex. Monoclonal and polyclonal anti-GPIb-IX complex antibodies inhibited to only a moderate degree (< 40%) the binding of the anti-FcγRII monoclonal antibody, IV.3, to platelets. In contrast, 6 of 12 anti-GPIIb-IIIa monoclonal antibodies and a polyclonal, affinity-purified rabbit anti-GPIIb-IIIa antibody strongly cross-blocked the binding of IV.3 to platelets. This inhibition was dependent upon the Fab-mediated binding of these antibodies to the GPIIb-IIIa complex since they did not inhibit the binding of IV.3 to Glanzmann's thrombasthenic platelets which have normal levels of FcγRII but lack the GPIIb-IIIa complex. The anti-GPIIb-IIIa monoclonal antibodies, AP3 and VM16a, had no effect on platelet aggregation induced by ADP or thrombin but inhibited Fc-receptor-dependent platelet aggregation as induced by either acetone-aggregated human IgG or by activating monoclonal antibodies against GPIV, PTA1 or CD9. F(ab')(2) fragments of these two anti-GPIIb-IIIa monoclonal antibodies also inhibited Fc-receptor-dependent platelet aggregation indicating that the observed interference by intact antibody was not due to the direct interaction of the Fc-portion of the antigen-antibody complex with FcγRII. In addition, the inhibitory anti-GPIIb-IIIa antibodies cross-blocked the binding of IV.3 to platelets at 0°C as well as at 22°C suggesting that the observed inhibition was not dependent on the lateral mobility of either GP IIb-IIIa or FcγRII in the platelet membrane. The combined results therefore strongly suggest that the platelet Fc-receptor, FcγRII, is topographically associated with the GPIIb-IIIa complex in the intact platelet membrane.