Michael C. Berndt

Heparin-induced thrombocytopenia: mechanism of interaction of the heparin-dependent antibody with platelets.

The interaction of the heparin‐dependent antibody with heparin and platelets has been studied using the sera and purified IgG of four patients with heparin‐induced thrombocytopenia. Both normal platelets and Bernard‐Soulier syndrome (BSS) platelets which lack glycoprotein (GP) Ib, GPV and GPIX, aggregated in response to patient serum or IgG, but only in the presence of heparin. A monoclonal antibody (Mab) against platelet Fc II receptor (IV. 3) strongly inhibited the heparin‐dependent aggregation of both normal and BSS platelets induced by patient sera/IgG. Inhibition by the anti‐GPIb Mab (AK2) was variable and occurred only with normal platelets. Anti‐GPIX Mab (FMC 25) was not inhibitory with either normal or BSS platelets. Similar results were obtained using 14C‐serotonin release instead of platelet aggregation as a measure of platelet activation. These findings suggest that (1) the reaction of the heparin‐dependent antibody with platelets and heparin is mediated by a Fc‐dependent mechanism, (2) GPIb, GPV and GPIX are not involved in this reaction, and (3) the inhibitory effect of anti‐GPIb Mab on normal platelets is due to steric interference consistent with the platelet Fc receptor being in close proximity to GPIb.

PSGL-1-MEDIATED ADHESION OF HUMAN HEMATOPOIETIC PROGENITORS TO P-SELECTIN RESULTS IN SUPPRESSION OF HEMATOPOIESIS

Cellular interactions are critical for the regulation of hematopoiesis. The sialomucin PSGL-1/CD162 mediates the attachment of mature leukocytes to P-selectin. We now show that PSGL-1 also functions as the sole receptor for P-selectin on primitive human CD34+ hematopoietic progenitor cells (HPC). More importantly, ligation of PSGL-1 by immobilized or soluble ligand or anti-PSGL-1 antibody results in a profound suppression of HPC proliferation stimulated by potent combinations of early acting hematopoietic growth factors. These data demonstrate an unanticipated but extremely marked growth-inhibitory effect of P-selectin on hematopoiesis and provide direct evidence that PSGL-1, in addition to its well-documented role as an adhesion molecule on mature leukocytes, is a potent negative regulator of human hematopoietic progenitors.

Characterization of human platelet GMP-140 as a heparin-binding protein

Human platelet GMP-140 has been identified as a heparin-binding protein. Purified platelet GMP-140 bound to Heparin-Sepharose CL-6B and was eluted by approximately 0.5 M sodium chloride. Radioiodinated GMP-140 bound specifically and saturably to heparin immobilized on Matrex-Pel 102 beads. Binding of radioiodinated GMP-140 to heparin-Matrex-Pel 102 beads was divalent cation-independent and was strongly inhibited by excess fluid phase GMP-140 and heparin and by other sulfated glycans such as fucoidin and dextran-sulfate. Binding was not inhibited by chondroitins 4- and 6-sulfate or mannose 6-phosphate.

Heparin-induced thrombocytopenia: effects of rabbit IgG, and its Fab and FC fragments on antibody-heparin-platelet interaction.

The mechanism whereby the antibody interacts with platelets and heparin is still unclear. Two mechanisms have been proposed previously. In this study, purified rabbit IgG and its Fab and Fc fragments were used to differentiate the two possible mechanisms

Detection of an epitope specific for the dissociated form of glycoprotein IIIa of platelet membrane glycoprotein IIb‐IIIa complex and its expression on the surface of adherent platelets

Summary Glycoproteins (GPs) IIb and IIIa form a Ca2+‐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 Glanzmanns 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 ± 103 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 ± 103 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 l25I‐CRC54 to adherent platelets in the presence of divalent cations was about 4 times higher than to platelets in suspension, 8.68 ± 0.48 ± 103 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.

The murine monoclonal antibody, 14A2.H1, identifies a novel platelet surface antigen.

Summary A murine monoclonal antibody 14A2.H1, raised against acute myeloid leukaemia cells, identifies a previously undescribed 27 kDa platelet surface glycoprotein which is expressed at low copy number (103/platelet). MAb 14A2.H1 caused aggregation of platelets which was dependent on FcγRII. Binding of the antibody to platelets was not altered by activation by thrombin or phorbol ester. In haemopoietic cell populations the antibody bound to megakaryocytes, monocytes (weakly), several myeloid leukaemic cell lines and fresh myeloid leukaemic blasts from some patients. Lymphocytes, lymphoid cell lines, neutrophils and haemopoietic progenitor cells were negative. Expression of the antigen was not restricted to haemopoietic cells as epithelial cells in tonsillar crypts and endothelial cells were positive.

The Amino Acid Sequence Glutamine-628 to Valine-646 Within the A1 Repeat Domain Mediates Binding of von Willebrand Factor to Bovine Brain Sulfatides and Equine Tendon Collagen

von Willebrand Factor (vWF) is a multifunctional glycoprotein in plasma and vascular subendothelial matrix which plays a major role in cellular adhesion. vWFdependent adhesion of platelets to the subendothelium at high shear rates involves a specific platelet membrane receptor, the glycoprotein (GP) Ib-IX complex. We have previously purified a 39/34-kiloDalton (kDa) dispase fragment of vWF (Leu-480/Val-481 to Gly-718) and demonstrated that this fragment contains the binding site for the GP Ib-IX complex [Andrews R K, et al. Biochemistry 1989; 28: 8326-83361. vWF also mediates agglutination of erythrocytes by a mechanism that appears to involve binding to membrane sulfatides. In this study, we demonstrate that the 39/34-kDa vWF fragment also contains an exclusive discrete binding domain for membrane sulfatides and that the sulfatide-binding sequence also mediates binding of vWF to equine tendon collagen. Specific binding of (125)I-vWF to sulfatides immobilized on microtiter wells was completely inhibited by unlabeled vWF (IC(50)∼0.02 μ;M) and by the isolated 39/34-kDa vWF fragment (IC(50)∼0.8 μ;M). A specific anti-39/34-kDa fragment rabbit polyclonal antibody, but not nonimmune immunoglobulin, also strongly inhibited the vWF-sulfatide interaction in this assay. Using synthetic peptides corresponding to hydrophilic sequences from within the 39/34-kDa vWF fragment, a positively-charged sequence, Gln-628 to Val-646, was identified as mediating specific binding of vWF to sulfatides, since it competitively inhibited this interaction (IC(50)∼0.6 μ;M) comparable on a molar basis to the 39/34-kDa vWF fragment (IC, -0.8 μ;M). The inhibition by the Gln-626 to Val-646 peptide was specific since neither other peptides from the 39/34-kDa domain of vWF nor another highly basic peptide, polylysine, at comparable concentrations to the Gln-628 to Val-646 peptide blocked vWF binding to sulfatides. Similarly, the Gln-628 to Val-646 peptide blocked binding of vWF to equine tendon type I collagen (IC(50) of 0.6 μ;M) suggesting that this interaction probably involves recognition of a sulfatide-like impurity in the collagen preparation. The specific binding of vWF to sulfatides via a discrete peptide sequence, Gln-628 to Val-646, within the A1 repeat domain suggests the potential for involvement of sulfatides as a class of receptors for vWF in cellular adhesion.

Topographical Association of the Platelet Fc-receptor with the Glycoprotein IIb-IIIa Complex

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 Glanzmanns 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.

Cisplatin‐induced platelet activation requires mononuclear cells: role of GMP‐140 and modulation of procoagulant activity

Cytotoxic drugs may potentiate the thrombotic complications in patients with malignancies and platelet function abnormalities have been reported after initiation of cisplatin therapy. This report describes a prolonged activation of platelets over 6‐24 h co‐culture with peripheral blood mononuclear cells (PBM) by pharmacological doses of cisplatin. Cisplatin had no direct effect on platelets and depended on PBM to produce aggregation which was apparently not mediated by products of the cyclooxygenase or lipoxygenase pathways, by platelet activation factor (PAF) or by thrombin. Although platelet aggregation normally involves the binding of fibrinogen to the β3 integrin, GP IIb‐IIIa, on activated platelets, the cisplatin‐dependent platelet aggregation observed in the co‐culture experiments was not inhibited by anti‐GP IIb‐IIIa monoclonal antibody which blocks fibrinogen‐dependent aggregation nor by an adhesive peptide containing the RGDS integrin recognition sequence. Rather, aggregation appeared to involve a novel 140 kD granule membrane protein (GMP‐140) mediated mechanism since aggregation was almost completely blocked by Fab fragments of antibody to GMP‐140 and was inhibited by fluid‐phase GMP‐140. At concentrations of cisplatin, adriamycin, and LPS that induced equivalent levels of tissue factor of blood monocytes, prothrombinase activity was significantly greater in cultures containing cisplatin. Prothrombinase activity was dependent on the presence of platelets and the rate of thrombin formation was enhanced by factor Xa generated by the tissue factor‐factor VIIa complex. These studies suggest that the vascular and thrombotic complications associated with cisplatin therapy are mediated, at least in part, by platelet activation and aggregation and monocyte procoagulant activity.

The Interaction of von Willebrand Factor and the Platelet Glycoprotein Ib-IX Complex

The interaction between blood platelets and the vessel wall represents the initial event in the haemostatic response to vessel injury. At a molecular level, this interaction involves the platelet membrane glycoprotein (GP) Ib-IX complex, von Willebrand factor (vWF) and a component (s) of the subendothelial matrix. In recent years, the primary sequences of both the vWF molecule and the three chains comprising the GP Ib-IX complex, i.e., GP Ib(α), GP Ib(β) and GP IX, have been determined, providing further insight into the structure-function relationships of these molecules. As a result, much recent investigation has been directed towards identifying small amino acid sequences in the primary chains of both GP Ib(α) and vWF which define the adhesive interaction. This review summarises current data concerning structure-function analysis of vWF and the GP Ib-IX complex and the putative identification of ligand- and receptor-binding domains involved in the interaction of vWF with the GP Ib-IX complex.